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Alternariol (AOH) and alternariol monomethyl ether (AME) are secondary metabolites produced by fungi belonging to the genus Alternaria, which generally contaminate fruits but also cereal crops and vegetables. The objective of this study was to investigate if the co-exposure of the swine epithelial intestinal cell line (IPEC-1) to a mixture of mycotoxins would cause an increase in toxicity as compared with exposure to a single toxin. The effects of individual toxins as well as those of their combination (1:1 ratio), in a range of 1–250 μM, were assessed in vitro for the cell viability of proliferating IPEC-1 cells and then on parameters related to the oxidative stress. Our results indicate that both AOH and AME significantly decreased the IPEC-1 cell viability, but the cytotoxicity induced by the AOH + AME combination was significantly higher than that induced by the exposure to the individual toxins. The main interaction type ranged from slight synergy for the AOH-AME combination affecting 25% of cell viability (CI = 0.88), which evolved into a synergistic effect for a higher level of cytotoxicity IL50 (CI = 0.41) and a strong synergistic effect at IL90 (CI = 0.10). In addition, we investigated the effects of two low concentrations (2.5 μM and 5 μM) of AOH and AME mycotoxins administered individually or in combination on oxidative stress in IPEC-1 cells. Both AOH and AME can induce an increase in reactive oxygen species—ROS (+) cells%—and oxidative damage in porcine IPEC-1 cells. At least an additive effect was observed when the cells were exposed to the combination of AOH-AME, consisting of an increase in the percentage of ROS (+) cells and the oxidation of lipids, proteins, and DNA as compared with the individual toxin effect. A breakdown of the antioxidant defense was observed in IPEC-1 cells after the exposure to individual toxins, related to the decrease in the activity of antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT), but no additive or synergic effect resulted after the exposure to the mixture of the toxins. In conclusion, our data indicate that both AOH and AME interfere with cell proliferation and oxidative stress. Moreover, the exposure of IPEC-1 cells to the combination of AOH and AME mycotoxins had a dose-dependent synergistic effect on IPEC-1 cell viability. Also, the oxidative damage induced in IPEC-1 cells by the combination of AOH and AME was stronger than the effects of individual toxins. However, the signaling pathways responsible for the toxicity of AOH, AME, and their combinations need further investigations in order to provide important data for risk assessments in swine in the case of the contamination of feed with Alternaria toxins. [ABSTRACT FROM AUTHOR]

Alternaria alternata is a prevalent postharvest pathogen that generates diverse mycotoxins, notably alternariol (AOH) and alternariol monomethyl ether (AME), which are recurrent severe contaminants. Nitrogen sources modulate fungal growth, development, and secondary metabolism, including mycotoxin production. The GATA transcription factor AreA regulates nitrogen source utilization. However, little is known about its involvement in the regulation of nitrogen utilization in A. alternata. To examine the regulatory mechanism of AaAreA on AOH and AME biosynthesis in A. alternata, we analyzed the impact of diverse nitrogen sources on the fungal growth, conidiation and mycotoxin production. The use of a secondary nitrogen source (NaNO3) enhanced mycelial elongation and sporulation more than the use of a primary source (NH4Cl). NaNO3 favored greater mycotoxin accumulation than did NH4Cl. The regulatory roles of AaAreA were further clarified through gene knockout. The absence of AaAreA led to an overall reduction in growth in minimal media containing any nitrogen source except NH4Cl. AaAreA positively regulates mycotoxin biosynthesis when both NH4Cl and NaNO3 are used as nitrogen sources. Subcellular localization analysis revealed abundant nuclear transport when NaNO3 was the sole nitrogen source. The regulatory pathway of AaAreA was systematically revealed through comprehensive transcriptomic analyses. The deletion of AaAreA significantly impedes the transcription of mycotoxin biosynthetic genes, including aohR, pksI and omtI. The interaction between AaAreA and aohR, a pathway-specific transcription factor gene, demonstrated that AaAreA binds to the aohR promoter sequence (5'-GGCTATGGAAA-3'), activating its transcription. The expressed AohR regulates the expression of downstream synthase genes in the cluster, ultimately impacting mycotoxin production. This study provides valuable information to further understand how AreA regulates AOH and AME biosynthesis in A. alternata, thereby enabling the effective design of control measures for mycotoxin contamination. [ABSTRACT FROM AUTHOR]

Alternariol (AOH), an emerging mycotoxin, inevitably exists widely in various food and feed commodities with cereals and fruits being particularly susceptible, raising global concerns over its harm to human and livestock health. The development of eco-friendly and efficient strategies to decontaminate AOH has been an urgent task. This study provided insight into the utilization of crude soybean hull peroxidase as a powerful biocatalyst for degrading AOH. The results confirmed that crude soybean hull peroxidase (SHP) could catalyze the oxidation of AOH by use of H2O2 as a co-substrate. The optimum reaction conditions for SHP-catalyzed AOH degradation were recorded at pH 4.0–8.0, at 42–57 °C, and at H2O2 concentration of 100–500 μM. Mass analysis elucidated the degradation of AOH through hydroxylation and methylation by crude SHP. Moreover, toxicological analysis indicated that crude SHP-catalyzed AOH degradation detoxified the hepatotoxicity of this mycotoxin. The performance of crude SHP to degrade AOH in food matrices was further evaluated, and it was found that the enzyme agent could achieve AOH degradation by 77% in wheat flour, 84% in corn flour, 34% in grape juice, and 26% in apple juice. Collectively, these findings establish crude SHP as a promising candidate for effective AOH degradation, with potential applications in the food and feed industry. [ABSTRACT FROM AUTHOR]

Alternaria species are mycotoxin-producing fungi known to infect fresh produce and to cause their spoilage. Humans get exposed to fungal secondary metabolites known as mycotoxin via the ingestion of contaminated food. Alternariol (AOH) (C14H10O5) is an isocoumarins produced by different species of Alternaria including Alternaria alternata. AOH is often found in grain, fruits and fruits-based food products with high levels in legumes, nuts, and tomatoes. AOH was first discovered in 1953, and it is nowadays linked to esophagus cancer and endocrine disruption due to its similarity to estrogen. Although considered as an emerging mycotoxin with no regulated levels in food, AOH occurs in highly consumed dietary products and has been detected in various masked forms, which adds to its occurrence. Therefore, this comprehensive review was developed to give an overview on recent literature in the field of AOH. The current study summarizes published data on occurrence levels of AOH in different food products in the last ten years and evaluates those levels in comparison to recommended levels by the regulating entities. Such surveillance facilitates the work of health risk assessors and highlights commodities that are most in need of AOH levels regulation. In addition, the effects of AOH on cells and animal models were summarized in two tables; data include the last two-year literature studies. The review addresses also the main characteristics of AOH and the possible human exposure routes, the populations at risk, and the effect of anthropogenic activities on the widespread of the mycotoxin. The commonly used detection and control methods described in the latest literature are also discussed to guide future researchers to focus on mitigating mycotoxins contamination in the food industry. This review aims mainly to serve as a guideline on AOH for mycotoxin regulation developers and health risk assessors. [ABSTRACT FROM AUTHOR]

Alternaria toxins are toxic metabolites of mold fungi of the genus Alternaria, which are widespread in nature. The purpose of the review was to characterize Alternaria toxins most frequently found in food products and posing a real threat to public health: alternariol (AOH) and its monomethyl ether (AME), altenuene (ALT), tentoxin (TEN), and tenuazonic acid (TeA). The existing toxicological data are insufficient to establish a value of provisional tolerable intake of Alternaria toxins. Based on the chemical structure the daily threshold of toxicological concern was determined: TeA and TEN, 1500 ng/kg b.w.; AOH and AME (taking into account their genotoxicity), 2.5 ng/kg b.w. Currently, the content of Alternaria toxins in food products is not regulated at the national or international levels. Liquid chromatography coupled to (tandem) mass spectrometry is the most preferred method of identification and quantification of Alternaria toxins. Research results indicate significant contamination with Alternaria toxins of food raw materials and products of their processing (including cereals and oilseeds crops; vegetables and fruits, spices, and baby food). Processing of raw materials contaminated with Alternaria toxins contributes, as a rule, to reducing their content in the ready-to-eat product, but does not allow for complete elimination of toxins. Children of the first three years of life are a population group under the greatest exposure to Alternaria toxins. At the same time, an intake of Alternaria toxins with a diet may exceed the threshold of toxicological concern and pose a real threat to health. The data presented in the review characterize Alternaria toxins as a significant risk factor for public health. To manage the corresponding risk, including through hygienic regulation, it is necessary to conduct additional research on the content of priority Alternaria toxins (AOH, AME, TeA, TEN, ALT) in food products, as well as clarify dose-dependent effects of their toxic action in order to minimize any possible adverse effects of Alternaria toxins on public health in the Russian Federation.

Post-harvest fruit rot caused by Alternaria species is one of the most important threats to the fruit industry. Post-harvest rot on sweet cherry (Prunus avium) fruit was observed in the fruit markets of the Haidian district of Beijing, China. The fungal isolates obtained from the infected sweet cherry fruits matched the descriptions of Alternaria alternata based on the morphology and multi-gene (ITS, endo-PG, and Alta1) sequence analysis. Pathogenicity tests indicated that ACT-3 was the most virulent isolate, exhibiting typical post-harvest fruit rot symptoms. Physiological studies revealed that the optimal conditions for the growth of ACT-3 were temperature of 28℃, water activity of 0.999, and pH of 8 with 87, 85, and 86mm radial growth of ACT-3 on a potato dextrose agar (PDA) medium, respectively, at 12days post-inoculation (dpi). Moreover, the fungus showed the highest growth on a Martin agar medium (MAM) modified (85mm) and a PDA medium (84mm) at 12 dpi. The proliferation of the fungus was visualized inside the fruit tissues by confocal and scanning electron microscope (SEM), revealing the invasion and destruction of fruit tissues. Alternaria mycotoxins, tenuazonic acid (TeA), and alternariol (AOH) were detected in five representative isolates by HPLC analysis. The highest concentrations of TeA (313 μg/mL) and AOH (8.9 μg/mL) were observed in ACT-6 and ACT-3 isolates, respectively. This study is the first to present a detailed report on the characteristics and proliferation of A. alternata associated with sweet cherry fruit rot and the detection of toxic metabolites. [ABSTRACT FROM AUTHOR]

Fungi of the genus Alternaria are ubiquitous plant pathogens and saprophytes which are able to grow under varying temperature and moisture conditions as well as on a large range of substrates. A spectrum of structurally diverse secondary metabolites with toxic potential has been identified, but occurrence and relative proportion of the different metabolites in complex mixtures depend on strain, substrate, and growth conditions. This review compiles the available knowledge on hazard identification and characterization of Alternaria toxins. Alternariol (AOH), its monomethylether AME and the perylene quinones altertoxin I (ATX-I), ATX-II, ATX-III, alterperylenol (ALP), and stemphyltoxin III (STTX-III) showed in vitro genotoxic and mutagenic properties. Of all identified Alternaria toxins, the epoxide-bearing analogs ATX-II, ATX-III, and STTX-III show the highest cytotoxic, genotoxic, and mutagenic potential in vitro. Under hormone-sensitive conditions, AOH and AME act as moderate xenoestrogens, but in silico modeling predicts further Alternaria toxins as potential estrogenic factors. Recent studies indicate also an immunosuppressive role of AOH and ATX-II; however, no data are available for the majority of Alternaria toxins. Overall, hazard characterization of Alternaria toxins focused, so far, primarily on the commercially available dibenzo-α-pyrones AOH and AME and tenuazonic acid (TeA). Limited data sets are available for altersetin (ALS), altenuene (ALT), and tentoxin (TEN). The occurrence and toxicological relevance of perylene quinone-based Alternaria toxins still remain to be fully elucidated. We identified data gaps on hazard identification and characterization crucial to improve risk assessment of Alternaria mycotoxins for consumers and occupationally exposed workers. [ABSTRACT FROM AUTHOR]

This study delved into the impact of two extrusion processing parameters—screw speed (SS at 400, 600, 800 RPM) and material moisture content in the extruder barrel (M at 12, 15, 18%) at constant feed rate (50 kg/h)—on reducing the content of alternariol (AOH), alternariol monomethyl ether (AME), tenuazonic acid (TeA), and tentoxin (TEN) in whole-grain red sorghum flour. Ultra-performance liquid chromatography combined with a triple-quadrupole mass spectrometer (UPLC-MS/MS) was employed for the determination of Alternaria toxin levels. The extruder die temperature fluctuated between 136 and 177 °C, with die pressures ranging from 0.16 to 6.23 MPa. The specific mechanical energy spanned from 83.5 to 152.3 kWh/t, the torque varied between 88 and 162.8 Nm, and the average material retention time in the barrel ranged from 5.6 to 13 s. The optimal parameters for reducing the concentration of all Alternaria toxins with a satisfactory quality of the sorghum snacks were: SS = 400 RPM, M = 12%, with a reduction of 61.4, 76.4, 12.1, and 50.8% for AOH, AME, TeA, and TEN, respectively. [ABSTRACT FROM AUTHOR]

Humans are constantly exposed to mixtures of different xenobiotics through their diet. One emerging concern is the Alternaria mycotoxin alternariol (AOH), which can occur in foods typically contaminated by the process contaminant acrylamide (AA). AA is a byproduct of the Maillard reaction produced in carbohydrate-rich foods during thermal processing. Given the genotoxic properties of AOH and AA as single compounds, as well as their potential co-occurrence in food, this study aimed to assess the cytotoxic, genotoxic, and mutagenic effects of these compounds in combination. Genotoxicity was assessed in HepG2 cells by quantifying the phosphorylation of the histone γ-H2AX, induced as a response to DNA double-strand breaks (DSBs). Mutagenicity was tested in Salmonella typhimurium strains TA98 and TA100 by applying the Ames microplate format test. Our results showed the ability of AOH and AA to induce DSBs and increase revertant numbers in S. typhimurium TA100, with AOH being more potent than AA. However, no synergistic effects were observed during the combined treatments. Notably, the results of the study suggest that the compounds exert mutagenic effects primarily through base pair substitutions. In summary, the data indicate no immediate cause for concern regarding synergistic health risks associated with the consumption of foods co-contaminated with AOH and AA. [ABSTRACT FROM AUTHOR]

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.

Alternariol (AOH) and alternariol monomethyl ether (AME) are secondary metabolites produced by fungi belonging to the genus Alternaria, which generally contaminate fruits but also cereal crops and vegetables. The objective of this study was to investigate if the co-exposure of the swine epithelial intestinal cell line (IPEC-1) to a mixture of mycotoxins would cause an increase in toxicity as compared with exposure to a single toxin. The effects of individual toxins as well as those of their combination (1:1 ratio), in a range of 1–250 μM, were assessed in vitro for the cell viability of proliferating IPEC-1 cells and then on parameters related to the oxidative stress. Our results indicate that both AOH and AME significantly decreased the IPEC-1 cell viability, but the cytotoxicity induced by the AOH + AME combination was significantly higher than that induced by the exposure to the individual toxins. The main interaction type ranged from slight synergy for the AOH-AME combination affecting 25% of cell viability (CI = 0.88), which evolved into a synergistic effect for a higher level of cytotoxicity IL50 (CI = 0.41) and a strong synergistic effect at IL90 (CI = 0.10). In addition, we investigated the effects of two low concentrations (2.5 μM and 5 μM) of AOH and AME mycotoxins administered individually or in combination on oxidative stress in IPEC-1 cells. Both AOH and AME can induce an increase in reactive oxygen species—ROS (+) cells%—and oxidative damage in porcine IPEC-1 cells. At least an additive effect was observed when the cells were exposed to the combination of AOH-AME, consisting of an increase in the percentage of ROS (+) cells and the oxidation of lipids, proteins, and DNA as compared with the individual toxin effect. A breakdown of the antioxidant defense was observed in IPEC-1 cells after the exposure to individual toxins, related to the decrease in the activity of antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT), but no additive or synergic effect resulted after the exposure to the mixture of the toxins. In conclusion, our data indicate that both AOH and AME interfere with cell proliferation and oxidative stress. Moreover, the exposure of IPEC-1 cells to the combination of AOH and AME mycotoxins had a dose-dependent synergistic effect on IPEC-1 cell viability. Also, the oxidative damage induced in IPEC-1 cells by the combination of AOH and AME was stronger than the effects of individual toxins. However, the signaling pathways responsible for the toxicity of AOH, AME, and their combinations need further investigations in order to provide important data for risk assessments in swine in the case of the contamination of feed with Alternaria toxins.

Alternariol (AOH), an emerging mycotoxin, inevitably exists widely in various food and feed commodities with cereals and fruits being particularly susceptible, raising global concerns over its harm to human and livestock health. The development of eco-friendly and efficient strategies to decontaminate AOH has been an urgent task. This study provided insight into the utilization of crude soybean hull peroxidase as a powerful biocatalyst for degrading AOH. The results confirmed that crude soybean hull peroxidase (SHP) could catalyze the oxidation of AOH by use of H2O2 as a co-substrate. The optimum reaction conditions for SHP-catalyzed AOH degradation were recorded at pH 4.0–8.0, at 42–57 °C, and at H2O2 concentration of 100–500 μM. Mass analysis elucidated the degradation of AOH through hydroxylation and methylation by crude SHP. Moreover, toxicological analysis indicated that crude SHP-catalyzed AOH degradation detoxified the hepatotoxicity of this mycotoxin. The performance of crude SHP to degrade AOH in food matrices was further evaluated, and it was found that the enzyme agent could achieve AOH degradation by 77% in wheat flour, 84% in corn flour, 34% in grape juice, and 26% in apple juice. Collectively, these findings establish crude SHP as a promising candidate for effective AOH degradation, with potential applications in the food and feed industry.

Post-harvest fruit rot caused by Alternaria species is one of the most important threats to the fruit industry. Post-harvest rot on sweet cherry (Prunus avium) fruit was observed in the fruit markets of the Haidian district of Beijing, China. The fungal isolates obtained from the infected sweet cherry fruits matched the descriptions of Alternaria alternata based on the morphology and multi-gene (ITS, endo-PG, and Alta1) sequence analysis. Pathogenicity tests indicated that ACT-3 was the most virulent isolate, exhibiting typical post-harvest fruit rot symptoms. Physiological studies revealed that the optimal conditions for the growth of ACT-3 were temperature of 28°C, water activity of 0.999, and pH of 8 with 87, 85, and 86 mm radial growth of ACT-3 on a potato dextrose agar (PDA) medium, respectively, at 12 days post-inoculation (dpi). Moreover, the fungus showed the highest growth on a Martin agar medium (MAM) modified (85 mm) and a PDA medium (84 mm) at 12 dpi. The proliferation of the fungus was visualized inside the fruit tissues by confocal and scanning electron microscope (SEM), revealing the invasion and destruction of fruit tissues. Alternaria mycotoxins, tenuazonic acid (TeA), and alternariol (AOH) were detected in five representative isolates by HPLC analysis. The highest concentrations of TeA (313 μg/mL) and AOH (8.9 μg/mL) were observed in ACT-6 and ACT-3 isolates, respectively. This study is the first to present a detailed report on the characteristics and proliferation of A. alternata associated with sweet cherry fruit rot and the detection of toxic metabolites.

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. [ABSTRACT FROM AUTHOR]

Mycotoxins are secondary metabolites of fungi that may affect both human and animal health. Some of them possess estrogenic activity, due to direct binding to estrogen receptors (ERs) and hence disturb the hormonal balance of the organism. Alternariol (AOH) was previously reported as genotoxic, estrogenic and immunomodulatory agent. However, detailed mechanism of its action has not been fully elucidated. Estrogen receptor α (ERα) was previously reported to modulate the proliferation and invasiveness of ovarian cancer cells. Thus, we decided to verify whether estrogenic-like mycotoxin may affect ovarian cancer cells via ERα. The results showed that AOH induces apoptosis and oxidative stress and that these effects are partially modulated by ERα. Moreover, AOH decreases the invasion and migration of ovarian cancer cells and promotes changes in the expression of genes and proteins that are associated with the invasiveness of cancer i.e. MMP9 , SNAIL1/2 , ZEB1/2 , VIM, CDH1 and CDH2. In conclusion, we postulate that AOH might significantly affect the viability and invasiveness of ovarian cancer cells via modulation of ERα and therefore possibly act as an endocrine disruptive agent in ovarian cancer cells. [Display omitted] • AOH decreases viability and induces oxidative stress and apoptosis in ovarian cancer cells. • ERα participates in AOH-induced toxicity in ovarian cancer cells. • AOH modulates the migration and the invasiveness of ovarian cancer cells via ERα. [ABSTRACT FROM AUTHOR]

Alternariol is a metabolite produced by Alternaria fungus that can contaminate a variety of food and feed materials. The objective of the present paper was to provide a prediction of Phase I and II metabolites of alternariol and a detailed ADME/Tox profile for alternariol and its metabolites using an in silico working model based on the MetaTox, SwissADME, pKCMS, and PASS online computational programs. A number of 12 metabolites were identified as corresponding to the metabolomic profile of alternariol. ADME profile for AOH and predicted metabolites indicated a moderate or high intestinal absorption probability but a low probability to penetrate the blood–brain barrier. In addition to cytotoxic, mutagenic, carcinogenic, and endocrine disruptor effects, the computational model has predicted other toxicological endpoints for the analyzed compounds, such as vascular toxicity, haemato-toxicity, diarrhea, and nephrotoxicity. AOH and its metabolites have been predicted to act as a substrate for different isoforms of phase I and II drug-metabolizing enzymes and to interact with the response to oxidative stress. In conclusion, in silico methods can represent a viable alternative to in vitro and in vivo tests for the prediction of mycotoxins metabolism and toxicity. [ABSTRACT FROM AUTHOR]

The mycotoxin alternariol (AOH) can be found in food products infected by Alternaria spp. and is considered an endocrine-disruptive mycotoxin. The main mechanism of AOH toxicity is associated with DNA damage and modulation of the inflammation process. Still, AOH is considered as one of the emerging mycotoxins. In this study, we have evaluated how AOH might affect the local steroidogenesis process in the prostate, in both normal and cancer cells. We have found that AOH itself modulates the cell cycle, inflammation, and apoptosis, rather than the steroidogenesis process in prostate cancer cells; however, in the presence of another steroidogenic agent, the influence on steroidogenesis is significant. Therefore, this is the first study to report the effect of AOH on local steroidogenesis in normal and prostate cancer cells. We postulate that AOH might modulate the release of the steroid hormones and expression of the key components by interfering with the steroidogenic pathway and might be considered a steroidogenesis-altering agent. [ABSTRACT FROM AUTHOR]

Alternariol is a toxic metabolite of Alternaria fungi and studies have shown multiple potential pharmacological effects. To outline the anticancer effects and mechanisms of alternariol and its derivatives based on database reports, an updated search of PubMed/MedLine, ScienceDirect, Web of Science, and Scopus databases was performed with relevant keywords for published articles. The studies found to suggest that this mycotoxin and/or its derivatives have potential anticancer effects in many pharmacological preclinical test systems. Scientific reports indicate that alternariol and/or its derivatives exhibit anticancer through several pathways, including cytotoxic, reactive oxygen species leading to oxidative stress and mitochondrial dysfunction-linked cytotoxic effect, anti-inflammatory, cell cycle arrest, apoptotic cell death, genotoxic and mutagenic, anti-proliferative, autophagy, and estrogenic and clastogenic mechanisms. In light of these results, alternariol may be one of the hopeful chemotherapeutic agents. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The Alternaria species are considered to produce a plethora of several mycotoxins constituting a risk factor for both human and animal health. This work aimed mainly to explore the cytotoxicity of a combined mixture of altenuene (ALT), alternariol (AOH), tenuazonic acid (TeA), and altenuisol (AS) toxins produced by pathogenic A. alternata toward human oral epithelial cells (PCS-200-014), lung fibroblast cells (WI-38), and male albino rats. The sequencing of the multi-locus, RNA polymerase second largest subunit (rpb2), glyceraldehyde-3-phosphate dehydrogenase (gapdh), and Alternaria major allergen gene (Alt a 1) was performed to infer relationships among isolated Alternaria species. The phylogenetic analysis of gapdh, rpb2, and Alt-a 1 sequence data indicated that all isolates resided in A. alternata. The pathogenic potentiality of A. alternata was investigated on tomato plants cv. super strain B under greenhouse conditions, and all isolates were pathogenic to tomato plants, with significant (p < 0.05) variations. The ability of A. alternata isolates to produce mycotoxins was also explored using high-performance liquid chromatography (HPLC). All tested isolates were able to produce at least one of the assessed mycotoxins--ALT, AOH, TeA, and AS--and ALT was reported as the dominant mycotoxin, produced by 80% of A. alternata isolates. The cytotoxic properties of the combined mixture of ALT, AOH, TeA, and AS at concentrations of 31.25, 62.50, 125, 250, and 500 µg/mL were assessed via the MTT assay method after exposure for 24 h versus the control. The treatment of both cell lines with combined mixtures of ALT, AOH, TeA, and AS showed a dose-dependent decrease in cell viability. The highest concentrations tested at 62.50, 125, 250, and 500 µg/mL significantly decreased cell viability and caused cell damage compared to the lowest concentration of 31.25 µg/mL and the control. The cytotoxicity and genotoxicity of the combined mixtures of ALT, AOH, TeA, and AS on male albino rats were also investigated via the gene expression of (TNF-α) and using hematological (CBC), chemical (alanine aminotransferase (ALT), aspartate aminotransferase (AST) and urea and creatinine), and histopathological analyses. A marked increase was observed in the levels of ALT, AST, urea and creatinine, TNF-α gene expression, red blood cells (RBCs), white blood cells (WBCs), hemoglobin (Hb), and packed cell volume % (PCV) after 28 days of exposure relative to the untreated control. Pathological alterations were also observed in the liver and kidney tissues of rats. Conclusively, this work provides a new understanding on the cytotoxicity and genotoxicity of mycotoxins of pathogenic A. alternata from tomatoes. [ABSTRACT FROM AUTHOR]

This study delved into the impact of two extrusion processing parameters—screw speed (SS at 400, 600, 800 RPM) and material moisture content in the extruder barrel (M at 12, 15, 18%) at constant feed rate (50 kg/h)—on reducing the content of alternariol (AOH), alternariol monomethyl ether (AME), tenuazonic acid (TeA), and tentoxin (TEN) in whole-grain red sorghum flour. Ultra-performance liquid chromatography combined with a triple-quadrupole mass spectrometer (UPLC-MS/MS) was employed for the determination of Alternaria toxin levels. The extruder die temperature fluctuated between 136 and 177 °C, with die pressures ranging from 0.16 to 6.23 MPa. The specific mechanical energy spanned from 83.5 to 152.3 kWh/t, the torque varied between 88 and 162.8 Nm, and the average material retention time in the barrel ranged from 5.6 to 13 s. The optimal parameters for reducing the concentration of all Alternaria toxins with a satisfactory quality of the sorghum snacks were: SS = 400 RPM, M = 12%, with a reduction of 61.4, 76.4, 12.1, and 50.8% for AOH, AME, TeA, and TEN, respectively.

Alternariol is a toxic metabolite of Alternaria fungi and studies have shown multiple potential pharmacological effects. To outline the anticancer effects and mechanisms of alternariol and its derivatives based on database reports, an updated search of PubMed/MedLine, ScienceDirect, Web of Science, and Scopus databases was performed with relevant keywords for published articles. The studies found to suggest that this mycotoxin and/or its derivatives have potential anticancer effects in many pharmacological preclinical test systems. Scientific reports indicate that alternariol and/or its derivatives exhibit anticancer through several pathways, including cytotoxic, reactive oxygen species leading to oxidative stress and mitochondrial dysfunction-linked cytotoxic effect, anti-inflammatory, cell cycle arrest, apoptotic cell death, genotoxic and mutagenic, anti-proliferative, autophagy, and estrogenic and clastogenic mechanisms. In light of these results, alternariol may be one of the hopeful chemotherapeutic agents.

Brown and black spots, caused by Stemphylium and Alternaria species, are important fungal diseases affecting European pear (Pyrus communis) in orchards. Both fungal genera cause similar symptoms, which could favor misidentification, but Alternaria spp. are increasingly reported due to the changing climatic conditions. In this study, Alternaria spp. were isolated from symptomatic leaves and fruits of European pear, and their pathogenicity was evaluated on pear fruits from cultivar Abate Fétel, and molecular and chemical characterization were performed. Based on maximum likelihood phylogenetic analysis, 15 of 46 isolates were identified as A. arborescens species complex (AASC), 27 as A. alternata, and four as Alternaria sp. Both species were isolated from mature fruits and leaves. In pathogenicity assays on pear fruits, all isolates reproduced the symptoms observed in the field, by both wound inoculation and direct penetration. All but one isolate produced Alternaria toxins on European pears, including tenuazonic acid and alternariol (89.1% of the isolates), alternariol monomethyl ether (89.1%), altertoxin I (80.4%), altenuene (50.0%), and tentoxin (2.2%). These isolates also produced at least two mycotoxins, and 43.5% produced four mycotoxins, with an average total concentration of the Alternaria toxins exceeding 7.58 × 106 ng/kg. Our data underline the potential risks for human health related to the high mycotoxin content found on fruits affected by black spot. This study also represents the first report of AASC as an agent of black spot on European pear in Italy. [ABSTRACT FROM AUTHOR]

Alternariol (AOH) is an emerging mycotoxin produced by Alternaria strains. The acute toxicity of the mycotoxin is low; however, chronic exposure to AOH may result in the development of endocrine disruptor and/or carcinogenic effects. The toxicokinetic properties of AOH have barely been characterized. Therefore, in this study, we aimed to investigate its interactions with CYP (1A2, 2C9, 2C19, 2D6, and 3A4) enzymes and OATP (1A2, 1B1, 1B3, and 2B1) transporters employing in vitro enzyme assays and OATP overexpressing cells, respectively. Our results demonstrated that AOH is a strong inhibitor of CYP1A2 (IC50 = 0.15 μM) and CYP2C9 (IC50 = 7.4 μM). Based on the AOH depletion assays in the presence of CYP enzymes, CYP1A2 is mainly involved, while CYP2C19 is moderately involved in the CYP-catalyzed biotransformation of the mycotoxin. AOH proved to be a strong inhibitor of each OATP transporter examined (IC50 = 1.9 to 5.4 μM). In addition, both direct and indirect assays suggest the involvement of OATP1B1 in the cellular uptake of the mycotoxin. These findings promote the deeper understanding of certain toxicokinetic interactions of AOH. [ABSTRACT FROM AUTHOR]

The breast cancer is most frequently diagnosed cancer in the women worldwide. Our study investigated the anticancer effect of alternariol, a secondary metabolite originated from endophytic fungi, against DMBA induced breast carcinoma on Wistar rats. The toxicity study investigated the LD50 and the subsequent doses of alternariol for the carcinogenic study. The breast cancer was developed in rats via induction of DMBA (5 mg/kg, i.v.) and the carcinogenic study was continued for 24 weeks. The induction of breast cancer and the chemotherapeutic effect of alternariol were assessed through histopathological analysis of rat mammary tissue, followed by immunohistochemical analysis, cell proliferation assay and apoptosis assay by TUNEL method. The result showed that alternariol therapy decreased the hyperplastic lesions of mammary tissue and restored the normal histopathological characteristics of breast tissue. Furthermore, alternariol treatment downregulated the expression of carcinogenic markers such as PI3K and Akt increased the expression of apoptotic markers including p53, caspase-3 and Bax. Alternariol therapy also decreased the cellular proliferation and enhanced the apoptotic events. In conclusion, the breast cancer progression was significantly reduced via induction of apoptotic events and inhibition of cell propagation which allowed constructing of suitable mechanism for alternariol mediated chemotherapeutic approach. [ABSTRACT FROM AUTHOR]

Humans are constantly exposed to mixtures of different xenobiotics through their diet. One emerging concern is the Alternaria mycotoxin alternariol (AOH), which can occur in foods typically contaminated by the process contaminant acrylamide (AA). AA is a byproduct of the Maillard reaction produced in carbohydrate-rich foods during thermal processing. Given the genotoxic properties of AOH and AA as single compounds, as well as their potential co-occurrence in food, this study aimed to assess the cytotoxic, genotoxic, and mutagenic effects of these compounds in combination. Genotoxicity was assessed in HepG2 cells by quantifying the phosphorylation of the histone γ-H2AX, induced as a response to DNA double-strand breaks (DSBs). Mutagenicity was tested in Salmonella typhimurium strains TA98 and TA100 by applying the Ames microplate format test. Our results showed the ability of AOH and AA to induce DSBs and increase revertant numbers in S. typhimurium TA100, with AOH being more potent than AA. However, no synergistic effects were observed during the combined treatments. Notably, the results of the study suggest that the compounds exert mutagenic effects primarily through base pair substitutions. In summary, the data indicate no immediate cause for concern regarding synergistic health risks associated with the consumption of foods co-contaminated with AOH and AA.

Chickpeas are a very important legume due to their nutritional richness and high protein content and they are used as food for humans and as fodder for livestock. However, they are susceptible to fungal infections and mycotoxin contamination. The Alternaria genus was among the main fungi isolated from chickpea samples in Argentina. The species within this genus are able to produce several mycotoxins such as alternariol (AOH), alternariol monomethyl ether (AME), and tenuazonic acid (TA). So, the objectives of this study were to identify the Alternaria spp. found in the chickpea samples and to determine their toxigenic potential in vitro. A phylogenetic analysis of 32 Alternaria strains was carried out based on the combined sequences of the tef1, gpd, and Alt a1 genes. All Alternaria strains clustered into the section Alternaria and were identified as A. alternata and A. arborescens. Further, the toxigenic profile of each strain was determined in a ground rice–corn steep liquor medium and analysed by HPLC. Most strains were able to co-produce AOH, AME, and TA. These results indicate a potential risk for human health when consuming chickpeas since this legume could be contaminated with Alternaria and its mycotoxins, which are not yet regulated in food. [ABSTRACT FROM AUTHOR]

Alternariol is a metabolite produced by Alternaria fungus that can contaminate a variety of food and feed materials. The objective of the present paper was to provide a prediction of Phase I and II metabolites of alternariol and a detailed ADME/Tox profile for alternariol and its metabolites using an in silico working model based on the MetaTox, SwissADME, pKCMS, and PASS online computational programs. A number of 12 metabolites were identified as corresponding to the metabolomic profile of alternariol. ADME profile for AOH and predicted metabolites indicated a moderate or high intestinal absorption probability but a low probability to penetrate the blood–brain barrier. In addition to cytotoxic, mutagenic, carcinogenic, and endocrine disruptor effects, the computational model has predicted other toxicological endpoints for the analyzed compounds, such as vascular toxicity, haemato-toxicity, diarrhea, and nephrotoxicity. AOH and its metabolites have been predicted to act as a substrate for different isoforms of phase I and II drug-metabolizing enzymes and to interact with the response to oxidative stress. In conclusion, in silico methods can represent a viable alternative to in vitro and in vivo tests for the prediction of mycotoxins metabolism and toxicity.

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Objectives: The lung cancer is most frequently diagnosed cancer incidence worldwide. A large number of populations are heavily affected to this every year with poor prognosis. Methods: Our study investigated the anticancer effect of alternariol, a secondary metabolite, on A549 lung cancer cell line and benzo-α-pyrene induced lung carcinoma model on balb/c mice. The cytotoxicity assay, DAPI staining and the flow cytometry was performed to assess the anticancer efficacy of alternariol in A549 lung cancer cell. For in vivo study the toxicity study was performed. The lung cancer was developed in the animals via intraperitoneal administration of benzo-α-pyrene and subsequently 2 weeks later alternariol treatment was carried out for 24 weeks. The chemotherapeutic effect of alternariol was assessed through histopathological analysis, followed by immunohistochemistry and in vivo antioxidant study. Results: The in vitro data suggested a significant percentage of early and late apoptotic events in A549 cells with G0/G1 phase arrest which ultimately caused apoptosis followed by alternariol therapy. The in vivo study showed that alternariol therapy decreased the pulmonary fibrosis and formation of granuloma in lung tissue and restored the normal histopathological characteristics of lung. Furthermore, alternariol treatment downregulated the expression of PI3K, Akt and inflammatory mediators such as IL-6, TNF-α and increased the expression of apoptotic markers, p53. Conclusion: In conclusion, the treatment with alternariol effectively decreased the progression of lung cancer through the inhibition of carcinogenic markers by reprogramming the p53/PI3K/Akt pathway and IL-6/TNF-α mediated cytokine signaling in mice. [ABSTRACT FROM AUTHOR]

Alternariol (AOH) is a metabolic product of Alternaria , which has various toxic effects on humans and animals and its pollution is increasingly being discovered in fruits, vegetables, and grains. At present, the detection of AOH is mainly achieved through high-performance liquid chromatography-mass spectrometry and immunological methods. However, there is currently a lack of fast detection methods for on-site use. Considering the unique advantages of immunological testing methods, this study developed an immunolateral chromatographic quantitative method for detecting AOH using quantum dot fluorescent nanospheres (QDFNs) as signal amplification materials. The detection range of AOH is 5.2–4798 μg/kg, 50 % inhibiting concentration (IC 50) is 156.4 μg/kg, limit of detection (LOD) value is 2.32 μg/kg, and limit of quantification (LOQ) value is 6.96 μg/kg. The recovery rates of AOH in tomato and strawberry were 95.27%–111.63% and 88.43%–96.22%, respectively. In brief, the developed approach has good accuracy and repeatability and can be used for the detection of AOH. • Quantum dot fluorescent nanosphere coupled with antibody was used as detection probe. • Established an immunological chromographic quantitative method for detection of AOH. • The detection range of method is 5.2–4798 μg/kg, with a limit of detection of 2.32 μg/kg. • Our designed test strips can serve as potential point-of-care testing platform for detection. [ABSTRACT FROM AUTHOR]

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.

A new polymorph of the mycotoxin alternariol is reported and characterized by single crystal X-ray diffraction. Structural data, Hirshfeld surface analysis, and 2D fingerprint plots are used to compare differences in the intermolecular interactions of the orthorhombic Pca21 Form I (previously reported) and the monoclinic P21/c Form II (herein reported). The polymorphs have small differences in planarity—7.55° and 2.19° between the terminal rings for Form I and Form II, respectively—that brings about significant differences in the crystal packing and O-H...H interactions. [ABSTRACT FROM AUTHOR]

Alternariol (AOH), alternariol monomethyl-ether (AME), and tenuazonic acid (TeA) are major mycotoxins produced by fungi of the genus Alternaria and are common contaminants of food products such as fruits, vegetables, cereals and grains. Alternaria mycotoxins are known to cause relevant economic losses and to have a negative impact on human and animal health. EFSA stated in its scientific opinion that data on the toxicity of Alternaria mycotoxins in humans and livestock are generally lacking, precluding proper hazard characterization. This study aimed to fill some knowledge gaps by studying the in vitro cytotoxicity toward human intestinal epithelial cells (Caco-2) and hepatocytes (HepG2). Cytotoxic properties were assessed by flow cytometric analyses of remaining viable cells (i.e., propidium iodide negative) after mycotoxin exposure for 24–48 h versus solvent control. Treatment of cells with single doses of AOH, AME, and TeA resulted in a dose-dependent loss of cell viability for both cell lines. Half maximal effective concentrations (EC50) of the different mycotoxins were comparable for the two cell lines. On HepG2 cells, EC50 values varying between 8 and 16, 4 and 5, and 40 and 95 μg/mL were calculated for AOH, AME, and TeA, respectively. On Caco-2 cells, EC50 values of 19 μg/mL and varying between 6 and 23, and 60 and 90 μg/mL were calculated for AOH, AME, and TeA, respectively. A general relative cytotoxicity ranking of about 1 = 1 >>> 3 was obtained for AOH, AME, and TeA, respectively. Treatment of both cell lines with combined binary and ternary mixtures of AOH, AME, and TeA in a 1:1:3 ratio, also showed a dose-dependent decrease in cell viability. For both cell lines, the binary combination of especially AME and TeA (1:3 ratio) but also of AOH and AME (1:1 ratio) significantly increased the cytotoxicity compared to the single compound toxicity, although mainly at the highest concentrations tested. The ternary combinations of AOH, AME, and TeA induced only a slight increase in cytotoxicity compared to the single mycotoxins, again at the highest concentrations tested. [ABSTRACT FROM AUTHOR]

Background: Endophytes are a rich source of bioactive natural products and suggested to contribute to the biological or defense activities of their host plants. Following our research on the discovery of bioactive metabolites from endophytes, Alternaria alternata was isolated from the leaves of Ficus carica L. fam Moraceae. Materials and Methods: Large scale cultivation of the endophytic strain was carried out and the obtained extract was subjected to preliminary screening of antifungal and cytotoxic activities. Results: Promising antifungal and cytotoxic activities were obtained for the extract. Bio-guided fractionation resulted in the isolation and identification of four alternariol derivatives (alternariol, alternariol-5-O-sulphate, alternariol-5-O-methyl ether, alternariol-5-O-methyl ether-4'-O-sulphate). The isolated compounds were tested for antifungal and cytotoxic effects. Results revealed highest antifungal activity for alternariol against A. terreus (MIC= 2.64 µg mL-1) and F. oxysporum (MIC= 36 µg mL-1) while alternariol-5-O-methyl ether exhibited the highest cytotoxicity against K-562 (CC50= 3.72 µg mL-1) and HUVEC (CC50= 2.06 µg mL-1) cell lines. Conclusion: All alternariol derivatives showed potent cytotoxic and antifungal activities against A. terreus suggesting the contribution of this endophyte in the known antimicrobial and anticancer activities of the host plant. [ABSTRACT FROM AUTHOR]

Alternariol (AOH) and alternariol monomethyl ether (AME) are two main Alternaria mycotoxins that endanger human health. In this study, a single-chain antibody fragment (scFv) capable of equivalently and specifically recognizing AOH and AME was first expressed, and its equivalent recognition mechanism was discussed. According to molecular docking and dynamic simulation, the C9 site, which was always exposed outside the binding cavity, made the structural differences between AOH and AME negligible. Due to the high similarity of structures, AOH and AME interacted with almost the same amino acids on the scFv; thus, the same interaction mode and interaction force were produced. This was considered to be the most critical reason for the equivalent recognition. Thus, the exposure of common structures was considered a potential strategy to obtain the equivalent recognition antibodies, and C9 was considered the key site in the process of hapten modification. These results laid a theoretical foundation for further research on antibodies against Alternaria mycotoxins. It could promote the rapid detection of AOH and AME in food and provide a new idea for targeted preparation of antibodies that could recognize multiple hazards with similar structures.

Alternariol (AOH), alternariol monomethyl-ether (AME), and tenuazonic acid (TeA) are major mycotoxins produced by fungi of the genus Alternaria and are common contaminants of food products such as fruits, vegetables, cereals and grains. Alternaria mycotoxins are known to cause relevant economic losses and to have a negative impact on human and animal health. EFSA stated in its scientific opinion that data on the toxicity of Alternaria mycotoxins in humans and livestock are generally lacking, precluding proper hazard characterization. This study aimed to fill some knowledge gaps by studying the in vitro cytotoxicity toward human intestinal epithelial cells (Caco-2) and hepatocytes (HepG2). Cytotoxic properties were assessed by flow cytometric analyses of remaining viable cells (i.e., propidium iodide negative) after mycotoxin exposure for 24–48 h versus solvent control. Treatment of cells with single doses of AOH, AME, and TeA resulted in a dose-dependent loss of cell viability for both cell lines. Half maximal effective concentrations (EC50) of the different mycotoxins were comparable for the two cell lines. On HepG2 cells, EC50 values varying between 8 and 16, 4 and 5, and 40 and 95 μg/mL were calculated for AOH, AME, and TeA, respectively. On Caco-2 cells, EC50 values of 19 μg/mL and varying between 6 and 23, and 60 and 90 μg/mL were calculated for AOH, AME, and TeA, respectively. A general relative cytotoxicity ranking of about 1 = 1 >>> 3 was obtained for AOH, AME, and TeA, respectively. Treatment of both cell lines with combined binary and ternary mixtures of AOH, AME, and TeA in a 1:1:3 ratio, also showed a dose-dependent decrease in cell viability. For both cell lines, the binary combination of especially AME and TeA (1:3 ratio) but also of AOH and AME (1:1 ratio) significantly increased the cytotoxicity compared to the single compound toxicity, although mainly at the highest concentrations tested. The ternary combinations of AOH, AME, and TeA induced only a slight increase in cytotoxicity compared to the single mycotoxins, again at the highest concentrations tested.

Alternaria alternata is a principal plant pathogen responsible for the biosynthesis of mycotoxins, including tenuazonic acid (TeA), alternariol (AOH), and alternariol monomethyl ether (AME). The velvet gene VeA is involved in fungal growth, development, and secondary metabolism, including mycotoxin biosynthesis via light regulation. In this study, the detailed regulatory roles of Aa VeA in A. alternata with various light sources were investigated from the comparative analyses between the wild type and the gene knockout strains. In fungal growth and conidiation, mycelial extension was independent of light regulation in A. alternata. Red light favored conidiation, but blue light repressed it. The absence of Aa VeA caused the marked reduction of hyphae extension and conidiophore formation even though red light could not induce more spores in ΔAa VeA mutant. The differentially expressed genes (DEGs) enriched in hyphal growth and conidiation were drastically transcribed from the comparatively transcriptomic profile between the wild type and ΔAa VeA mutant strains with or without light. In mycotoxin production, TeA biosynthesis seems no obvious effect by light regulation, but AOH and AME formation was significantly stimulated by blue light. Nevertheless, the disruption of Aa VeA resulted in a marked decrease in mycotoxin production and the action of the stimulation was lost via blue light for the abundant accumulation of AOH and AME in the ΔAa VeA strain. From DEG expression and further verification by RT-qPCR, the loss of Aa VeA caused the discontinuous supply of the substrates for mycotoxin biosynthesis and the drastic decline of biosynthetic gene expression. In addition, pathogenicity depends on Aa VeA regulation in tomato infected by A. alternata in vivo. These findings provide a distinct understanding of the roles of Aa VeA in fungal growth, development, mycotoxin biosynthesis, and pathogenicity in response to various light sources. [ABSTRACT FROM AUTHOR]

The breast cancer is most frequently diagnosed cancer in the women worldwide. Our study investigated the anticancer effect of alternariol, a secondary metabolite originated from endophytic fungi, against DMBA induced breast carcinoma on Wistar rats. The toxicity study investigated the LD50 and the subsequent doses of alternariol for the carcinogenic study. The breast cancer was developed in rats via induction of DMBA (5 mg/kg, i.v.) and the carcinogenic study was continued for 24 weeks. The induction of breast cancer and the chemotherapeutic effect of alternariol were assessed through histopathological analysis of rat mammary tissue, followed by immunohistochemical analysis, cell proliferation assay and apoptosis assay by TUNEL method. The result showed that alternariol therapy decreased the hyperplastic lesions of mammary tissue and restored the normal histopathological characteristics of breast tissue. Furthermore, alternariol treatment downregulated the expression of carcinogenic markers such as PI3K and Akt increased the expression of apoptotic markers including p53, caspase-3 and Bax. Alternariol therapy also decreased the cellular proliferation and enhanced the apoptotic events. In conclusion, the breast cancer progression was significantly reduced via induction of apoptotic events and inhibition of cell propagation which allowed constructing of suitable mechanism for alternariol mediated chemotherapeutic approach. [ABSTRACT FROM AUTHOR]

Objectives: The lung cancer is most frequently diagnosed cancer incidence worldwide. A large number of populations are heavily affected to this every year with poor prognosis. Methods: Our study investigated the anticancer effect of alternariol, a secondary metabolite, on A549 lung cancer cell line and benzo-a-pyrene induced lung carcinoma model on balb/c mice. The cytotoxicity assay, DAPI staining and the flow cytometry was performed to assess the anticancer efficacy of alternariol in A549 lung cancer cell. For in vivo study the toxicity study was performed. The lung cancer was developed in the animals via intraperitoneal administration of benzo-α-pyrene and subsequently 2weeks later alternariol treatmentwas carried out for 24weeks. The chemotherapeutic effect of alternariolwas assessed through histopathological analysis, followed by immunohistochemistry and in vivo antioxidant study. Results: The in vitro data suggested a significant percentage of early and late apoptotic events in A549 cells with G0/G1 phase arrest which ultimately caused apoptosis followed by alternariol therapy. The in vivo study showed that alternariol therapy decreased the pulmonary fibrosis and formation of granuloma in lung tissue and restored the normal histopathological characteristics of lung. Furthermore, alternariol treatment downregulated the expression of PI3K, Akt and inflammatory mediators such as IL-6, TNF-α and increased the expression of apoptotic markers, p53. Conclusion: In conclusion, the treatment with alternariol effectively decreased the progression of lung cancer through the inhibition of carcinogenic markers by reprogramming the p53/PI3K/Akt pathway and IL-6/TNF-α mediated cytokine signaling inmice. [ABSTRACT FROM AUTHOR]

Alternariol (AOH) and ochratoxin A (OTA), two mycotoxins found in many foods worldwide, exhibit cytotoxicity and embryotoxicity, triggering apoptosis and cell cycle arrest in several mammalian cells and mouse embryos. The absorption rate of AOH from dietary foodstuff is low, meaning that the amount of AOH obtained from the diet rarely approaches the cytotoxic threshold. Thus, the potential harm of dietary consumption of AOH is generally neglected. However, previous findings from our group and others led us to question whether a low dosage of AOH could aggravate the cytotoxicity of other mycotoxins. In the present study, we examined how low dosages of AOH affected OTA-triggered apoptosis and embryotoxicity and investigated the underlying regulatory mechanism in mouse blastocysts. Our results revealed that non-cytotoxic concentrations of AOH (1 and 2 μM) could enhance OTA (8 μM)-triggered apoptotic processes and embryotoxicity in mouse blastocysts. We also found that AOH can enhance OTA-evoked intracellular reactive oxygen species (ROS) generation and that this could be prevented by pretreatment with the potent ROS scavenger, N-acetylcysteine. Finally, we observed that this ROS generation acts as a key inducer of caspase-dependent apoptotic processes and subsequent impairments of embryo implantation and pre- and post-implantation embryonic development. In sum, our results show that non-cytotoxic dosages of AOH can aggravate OTA-triggered apoptosis and embryotoxicity through ROS- and caspase-dependent signaling pathways. Graphical Abstract [ABSTRACT FROM AUTHOR]

Alternaria alternata is a principal plant pathogen responsible for the biosynthesis of mycotoxins, including tenuazonic acid (TeA), alternariol (AOH), and alternariol monomethyl ether (AME). The velvet gene VeA is involved in fungal growth, development, and secondary metabolism, including mycotoxin biosynthesis via light regulation. In this study, the detailed regulatory roles of AaVeA in A. alternata with various light sources were investigated from the comparative analyses between the wild type and the gene knockout strains. In fungal growth and conidiation, mycelial extension was independent of light regulation in A. alternata. Red light favored conidiation, but blue light repressed it. The absence of AaVeA caused the marked reduction of hyphae extension and conidiophore formation even though red light could not induce more spores in ΔAaVeA mutant. The differentially expressed genes (DEGs) enriched in hyphal growth and conidiation were drastically transcribed from the comparatively transcriptomic profile between the wild type and ΔAaVeA mutant strains with or without light. In mycotoxin production, TeA biosynthesis seems no obvious effect by light regulation, but AOH and AME formation was significantly stimulated by blue light. Nevertheless, the disruption of AaVeA resulted in a marked decrease in mycotoxin production and the action of the stimulation was lost via blue light for the abundant accumulation of AOH and AME in the ΔAaVeA strain. From DEG expression and further verification by RT-qPCR, the loss of AaVeA caused the discontinuous supply of the substrates for mycotoxin biosynthesis and the drastic decline of biosynthetic gene expression. In addition, pathogenicity depends on AaVeA regulation in tomato infected by A. alternata in vivo. These findings provide a distinct understanding of the roles of AaVeA in fungal growth, development, mycotoxin biosynthesis, and pathogenicity in response to various light sources.

The Alternaria species are considered to produce a plethora of several mycotoxins constituting a risk factor for both human and animal health. This work aimed mainly to explore the cytotoxicity of a combined mixture of altenuene (ALT), alternariol (AOH), tenuazonic acid (TeA), and altenuisol (AS) toxins produced by pathogenic A. alternata toward human oral epithelial cells (PCS-200-014), lung fibroblast cells (WI-38), and male albino rats. The sequencing of the multi-locus, RNA polymerase second largest subunit (rpb2), glyceraldehyde-3-phosphate dehydrogenase (gapdh), and Alternaria major allergen gene (Alt a 1) was performed to infer relationships among isolated Alternaria species. The phylogenetic analysis of gapdh, rpb2, and Alt-a 1 sequence data indicated that all isolates resided in A. alternata. The pathogenic potentiality of A. alternata was investigated on tomato plants cv. super strain B under greenhouse conditions, and all isolates were pathogenic to tomato plants, with significant (p < 0.05) variations. The ability of A. alternata isolates to produce mycotoxins was also explored using high-performance liquid chromatography (HPLC). All tested isolates were able to produce at least one of the assessed mycotoxins—ALT, AOH, TeA, and AS—and ALT was reported as the dominant mycotoxin, produced by 80% of A. alternata isolates. The cytotoxic properties of the combined mixture of ALT, AOH, TeA, and AS at concentrations of 31.25, 62.50, 125, 250, and 500 µg/mL were assessed via the MTT assay method after exposure for 24 h versus the control. The treatment of both cell lines with combined mixtures of ALT, AOH, TeA, and AS showed a dose-dependent decrease in cell viability. The highest concentrations tested at 62.50, 125, 250, and 500 µg/mL significantly decreased cell viability and caused cell damage compared to the lowest concentration of 31.25 µg/mL and the control. The cytotoxicity and genotoxicity of the combined mixtures of ALT, AOH, TeA, and AS on male albino rats were also investigated via the gene expression of (TNF-α) and using hematological (CBC), chemical (alanine aminotransferase (ALT), aspartate aminotransferase (AST) and urea and creatinine), and histopathological analyses. A marked increase was observed in the levels of ALT, AST, urea and creatinine, TNF-α gene expression, red blood cells (RBCs), white blood cells (WBCs), hemoglobin (Hb), and packed cell volume % (PCV) after 28 days of exposure relative to the untreated control. Pathological alterations were also observed in the liver and kidney tissues of rats. Conclusively, this work provides a new understanding on the cytotoxicity and genotoxicity of mycotoxins of pathogenic A. alternata from tomatoes.

Alternariol (AOH) is an emerging mycotoxin produced by Alternaria strains. The acute toxicity of the mycotoxin is low; however, chronic exposure to AOH may result in the development of endocrine disruptor and/or carcinogenic effects. The toxicokinetic properties of AOH have barely been characterized. Therefore, in this study, we aimed to investigate its interactions with CYP (1A2, 2C9, 2C19, 2D6, and 3A4) enzymes and OATP (1A2, 1B1, 1B3, and 2B1) transporters employing in vitro enzyme assays and OATP overexpressing cells, respectively. Our results demonstrated that AOH is a strong inhibitor of CYP1A2 (IC50 = 0.15 μM) and CYP2C9 (IC50 = 7.4 μM). Based on the AOH depletion assays in the presence of CYP enzymes, CYP1A2 is mainly involved, while CYP2C19 is moderately involved in the CYP-catalyzed biotransformation of the mycotoxin. AOH proved to be a strong inhibitor of each OATP transporter examined (IC50 = 1.9 to 5.4 μM). In addition, both direct and indirect assays suggest the involvement of OATP1B1 in the cellular uptake of the mycotoxin. These findings promote the deeper understanding of certain toxicokinetic interactions of AOH.

Using 126 endogenous lichen fungus (ELF) extracts, inhibitory activities against monoamine oxidases (MAOs) and cholinesterases (ChEs) were evaluated. Among them, extract ELF29 of the endogenous fungus Diaporthe mahothocarpus of the lichen Cladonia symphycarpia showed the highest inhibitory activity against hMAO-A. Compounds alternariol (AT), 50 -hydroxy-alternariol (HAT), and mycoepoxydiene (MED), isolated from the extract, had potent inhibitory activities against hMAOA with IC50 values of 0.020, 0.31, and 8.68 µM, respectively. AT, HAT, and MED are reversible competitive inhibitors of hMAO-A with Ki values of 0.0075, 0.116, and 3.76 µM, respectively. The molecular docking studies suggested that AT, HAT, and MED had higher binding affinities for hMAOA (−9.1, −6.9, and −5.6 kcal/mol, respectively) than for hMAO-B (−6.3, −5.2, and −3.7 kcal/mol, respectively). The relative tight binding might result from a hydrogen bond interaction of the three compounds with a Tyr444 residue in hMAO-A, whereas no hydrogen bond interaction was proposed in hMAO-B. In silico pharmacokinetics, the three compounds showed high gastrointestinal absorption without violating Lipinski’s five rules, but only MED showed high probability to cross the blood–brain barrier. These results suggest that AT, HAT, and MED are candidates for treating neuropsychiatric disorders, such as depression and cardiovascular disease. [ABSTRACT FROM AUTHOR]

Chickpeas are a very important legume due to their nutritional richness and high protein content and they are used as food for humans and as fodder for livestock. However, they are susceptible to fungal infections and mycotoxin contamination. The Alternaria genus was among the main fungi isolated from chickpea samples in Argentina. The species within this genus are able to produce several mycotoxins such as alternariol (AOH), alternariol monomethyl ether (AME), and tenuazonic acid (TA). So, the objectives of this study were to identify the Alternaria spp. found in the chickpea samples and to determine their toxigenic potential in vitro. A phylogenetic analysis of 32 Alternaria strains was carried out based on the combined sequences of the tef1, gpd, and Alt a1 genes. All Alternaria strains clustered into the section Alternaria and were identified as A. alternata and A. arborescens. Further, the toxigenic profile of each strain was determined in a ground rice–corn steep liquor medium and analysed by HPLC. Most strains were able to co-produce AOH, AME, and TA. These results indicate a potential risk for human health when consuming chickpeas since this legume could be contaminated with Alternaria and its mycotoxins, which are not yet regulated in food.

Glycyrrhiza glabra root (licorice) is a widely used herb for its beneficial effects on health. This study explored the protective effects of licorice extract against oxidative stress and testicular dysfunction caused by methotrexate (MTX). Mice were allocated into (i) negative control group that received saline; (ii) licorice extract group, orally administered with 200 mg/kg body weight (bw) licorice extract for 12 days; (iii) positive MTX-intoxicated group, injected with a single intraperitoneal dose of MTX (20 mg/kg bw) on day 7; and (iv) a protective group that received licorice extract for 12 days and then MTX on day 7 as in groups 2 and 3. Total proteins, albumin, globulins, malondialdehyde, glutathione peroxidase, reduced glutathione, IL-1, and IL-6 were measured in blood and testis samples collected from all groups. Testicular oxidative stress, serum reproductive hormones, and spermogram were examined. The expression of steroidogenesis-associated genes (translocator protein; and P450scc) was examined by quantitative real-time PCR. Bcl-2-associated X protein and cyclogenase-2 genes were examined by immunohistochemical analysis. The bioactive contents of licorice extract were confirmed by gas chromatography-mass spectrometry analysis. Pretreatment with licorice extract ameliorated the toxic effects of MTX on total proteins, albumin, and globulins and oxidative stress biomarkers and reversed the effect of MTX on examined serum and tissue antioxidants. Besides, MTX down-regulated mRNA expression of translocator protein and P450scc genes. Licorice extract averted the decrease in serum testosterone and the increase in IL-1β and IL-6 levels induced by MTX. Moreover, MTX increased sperm abnormalities and percentage of dead sperms and reduced sperm motility. These changes were absent in the licorice preadministered group. Licorice prevented the increase in immunoreactivity of testis for Bcl-2-associated X protein and cyclogenase-2 that were overexpressed in MTX-injected mice. Licorice extracts positively regulated the expression of steroidogenesis genes suppressed by MTX, increased antioxidant enzymes (glutathione peroxidase, reduced glutathione, and catalase) and reduced biomarker of oxidative stress (testicular malondialdehyde) and inflammatory cytokines (IL-1 and -6). Moreover, reduction in testicular tissue immunoreactivity to Bcl-2-associated X protein and cyclogenase-2. In conclusion, licorice extract mitigated the toxic effects of MTX-induced testicular dysfunction at biochemical, molecular, and cellular levels. [ABSTRACT FROM AUTHOR]