Search

The protection and promotion of agricultural niche products can be supported by the application of analytical techniques able to link food to its territory. This study aimed at exploring the possibility to discriminate between cereal samples from South Tyrol (Italy) and the neighboring regions (Trentino, East Tyrol, and North Tyrol) by their

Alternaria molds can produce a variety of different mycotoxins, often resulting in food contamination with chemical mixtures, posing a challenge for risk assessment. Some of these metabolites possess estrogenic properties, an effect whose toxicological relevance is questioned in the light of the strong genotoxic and cytotoxic properties of co-occurring toxins. Thus, we tested a complex extract from A. alternata for estrogenic properties in Ishikawa cells. By assessing alkaline phosphatase activity, we did not observe estrogen receptor (ER) activation at non-cytotoxic concentrations (≤ 10 µg/ml). Furthermore, an extract stripped of highly genotoxic perylene quinones also did not mediate estrogenic effects, despite diminished genotoxic properties in the comet assay (≥ 10 µg/ml). Interestingly, both extracts impaired the estrogenicity of 17β-estradiol (E2) at non-cytotoxic concentrations (5–10 µg/ml), indicating anti-estrogenic effects which could not be explained by the presence of known mycoestrogens. A mechanism for this unexpected result might be the activation of the aryl hydrocarbon receptor (AhR) by Alternaria metabolites, as indicated by the induction of CYP1A1 transcription. While a direct influence on the metabolism of E2 could not be confirmed by LC–MS/MS, literature describing a direct interplay of the AhR with estrogenic pathways points to a corresponding mode of action. Taken together, the present study indicates AhR-mediated anti-estrogenic effects as a novel mechanism of naturally co-occurring Alternaria toxin mixtures. Furthermore, our results confirm their genotoxic activity and raise questions about the contribution of still undiscovered metabolites to toxicological properties.

Fungal secondary metabolites frequently contaminate agricultural products implying a potential toxicological concern for consumers. We report a food survey investigating 17 Alternaria mycotoxins in tomato sauce, sunflower seed oil and wheat flour. Matrix-dependent median concentrations of the most frequently studied toxins, namely alternariol (AOH), alternariol monomethyl ether (AME), tenuazonic acid, and tentoxin ranged between 1.2 and 6.6, 0.5–1.3, 66–161, and 0.1–0.5 ng/g. Additionally, the modified toxins AOH-3-sulfate (up to 2.1 ng/g) and AME-3-sulfate (up to 17.5 ng/g) were detected in 9% and 34% of tomato sauces accounting for 7–100% of their parent toxin concentrations. The barely studied perylene quinones altertoxin I and alterperylenol were determined in all matrices with concentrations of up to 6 and 48 ng/g, respectively. The contamination patterns of sample subgroups were compared considering multiple factors (e.g. country of origin, type of agriculture, vendor, food processing). Samples from organic agriculture showed comparably higher median and maximum concentrations of the investigated Alternaria toxins, but also national and vendor-specific (supermarket chains vs. online stores) quality requirements for agricultural crops and food processing in the case of sunflower seed oils seem to affect the Alternaria toxin contamination patterns in food commodities. Overall, the presented study contributes comprehensive Alternaria toxin occurrence data which is necessary for future risk assessments evaluating potentially caused health concerns for consumers.

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

Breast milk substitutes, e.g. infant formulae, are commonly introduced to the diet within the first months of life. As the infants detoxifying capability is not fully developed, it is of vital importance to minimize their exposure to food contaminants. Here, we present a comprehensive multi-mycotoxin assessment in infant food. Samples from the Austrian and Czech market (n=59) were screened for 46 mycotoxins and key metabolites using two complementary LC-MS/MS methods. Trace levels of 17 mycotoxins, including aflatoxin B1 (0.4 µg/kg), zearalenone (1 (1 for infant foods. Interestingly, two toxins not reported previously, namely aflatoxicol and sterigmatocystin, were identified in 3 and 17% of infant foods, respectively.

Molds of the genus Alternaria produce several mycotoxins, some of which may pose a threat for health due to their genotoxicity. Due to the lack of adequate toxicological and occurrence data, they are currently not regulated. Interactions between mycotoxins, gut microbiota and food constituents might occur after food ingestion, modifying the bioavailability and, therefore, overall toxicity of mycotoxins. The present work aimed to investigate the impact of in vitro short-term fecal incubation on the in vitro DNA-damaging effects exerted by 5 µg/mL of an Alternaria alternata extract, containing, among others, 15 nM alternariol, 12 nM alternariol monomethyl ether, 241 nM altertoxin II and 301 nM stemphyltoxin III, all of which are known as genotoxic. The involvement of microorganisms, undigested food constituents and soluble substances of human fecal samples in modifying the composition and the genotoxicity of the extract was investigated through the application of LC–MS/MS analysis and comet assays in HT-29 cells. Results showed that the potential of the mycotoxins to induce DNA strand breaks was almost completely quenched, even before anaerobic incubation, by contact with the different fractions of the fecal samples, while the potency to induce formamidopyrimidine DNA glycosylase (FPG)-sensitive sites was only slightly reduced. These effects were in line with a reduction of mycotoxin concentrations found in samples analyzed by LC–MS/MS. Although a direct correlation between the metabolic activity of the gut microbiota and modifications in mycotoxin contents was not clearly observed, adsorptive phenomena to bacterial cells and to undigested food constituents might explain the observed modifications. Electronic supplementary material The online version of this article (10.1007/s00204-020-02831-1) contains supplementary material, which is available to authorized users.

Despite the frequent infection of agricultural crops by Alternaria spp., their toxic secondary metabolites and potential food contaminants lack comprehensive metabolic characterization. In this study, we investigated their bioavailability, metabolism, and excretion in vivo. A complex Alternaria culture extract (50 mg/kg body weight) containing 11 known toxins and the isolated lead toxin altertoxin II (0.7 mg/kg body weight) were administered per gavage to groups of 14 Sprague Dawley rats each. After 3 h and 24 h, plasma, urine and feces were collected to determine toxin recoveries. For reliable quantitation, an LC–MS/MS method for the simultaneous detection of 20 Alternaria toxins and metabolites was developed and optimized for either biological matrix. The obtained results demonstrated efficient excretion of alternariol (AOH) and its monomethyl ether (AME) via feces (> 89%) and urine (> 2.6%) after 24 h, while the majority of tenuazonic acid was recovered in urine (20 and 87% after 3 and 24 h, respectively). Moreover, modified forms of AOH and AME were identified in urine and fecal samples confirming both, mammalian phase-I (4-hydroxy-AOH) and phase-II (sulfates) biotransformation in vivo. Despite the comparably high doses, perylene quinones were recovered only at very low levels (altertoxin I, alterperylenol, Alternaria mycotoxins and their relevance on food safety.

The emerging Alternaria mycotoxin altertoxin II demonstrated substantial genotoxicity in vitro. Ubiquitous Alternaria ssp. frequently infest various agricultural crops, leading to economic losses and also potential food safety issues caused by associated mycotoxin contaminations. Due to the lack of commercially available reference standards, data on the general chemical behavior, the occurrence and the biological/toxicological effects of altertoxin II are scarce. Since tomatoes are particularly prone to Alternaria infestations, we simulated the storage and food processing of intact tomatoes and purees after altertoxin II-addition. We observed significant decrease in altertoxin II concentrations during storage at room temperature and particularly under thermal stress, by employing a validated LC-MS/MS method. Moreover, the reduction to the compound’s epoxide group to the alcohol, i.e. the formation of altertoxin I, was determined at considerable ratios in intact tomato fruits suggesting effective enzymatic xenobiotic metabolism.

Among various agricultural crops, tomatoes are particularly prone to Alternaria infections, which are frequently resulting in economic losses and mycotoxin contamination. To investigate potential health concerns implied for consumers, we simulated the storage and food processing steps of intact and blended tomatoes after addition of the highly genotoxic secondary metabolite altertoxin II. We observed a significant decrease in altertoxin II concentrations in samples stored at room temperature and particularly those undergoing thermal treatment by employing a validated LC MS/MS method. When kept at room temperature, 87-90 % of ATX II was recovered after 1.5 h in raw tomato purees and purees heated before ATX-II addition, and 47-49 % were recovered after 24 h. In intact tomato fruits the recovery was 23 % after 1.5 h and

The emerging Alternaria mycotoxin altertoxin II demonstrated substantial genotoxicity in vitro. Ubiquitous Alternaria ssp. frequently infest various agricultural crops, leading to economic losses and also potential food safety issues caused by associated mycotoxin contaminations. Due to the lack of commercially available reference standards, data on the general chemical behavior, the occurrence and the biological/toxicological effects of altertoxin II are scarce. Since tomatoes are particularly prone to Alternaria infestations, we simulated the storage and food processing of intact tomatoes and purees after altertoxin II-addition. We observed significant decrease in altertoxin II concentrations during storage at room temperature and particularly under thermal stress, by employing a validated LC-MS/MS method. Moreover, the reduction to the compound’s epoxide group to the alcohol, i.e. the formation of altertoxin I, was determined at considerable ratios in intact tomato fruits suggesting effective enzymatic xenobiotic metabolism.

The sulfated forms of the Fusarium toxin deoxynivalenol (DON), deoxynivalenol-3-sulfate (DON-3-Sulf) and deoxynivalenol-15-sulfate (DON-15-Sulf) were recently described, however little is known about their mechanism of action in mammalian cells. DON-3-Sulf and DON-15-Sulf were taken up by HT-29 colon carcinoma cells, although to a lesser extent compared to DON. All three compounds were found to enhance the intracellular ROS level in the dichlorofluorescein assay (≥ 1μM), even though substantial differences were observed in their cytotoxic potential. In silico modelling highlighted that DON-sulfates do not share the classical mechanism of action of DON, being unable to fit into the ribosomal pocket and trigger the classical ribotoxic stress response. However, DON-3-Sulf and DON-15-Sulf sustained a distinctive proliferative stimulus in HT-29 and activated autophagy. The mechanisms of action of DON-3-Sulf and DON-15-Sulf suggest a potential interplay between the onset of ribosomal inhibition and autophagy activation as an alternative and/or complementary mode of action for DON and its sulfated analogues.

Mycotoxins produced by Alternaria fungi are ubiquitous food contaminants, but analytical methods for generating comprehensive exposure data are rare. We describe the development of an LC-MS/MS method covering 17 toxins for investigating the natural occurrence of free and modified Alternaria toxins in tomato sauce, sunflower seed oil, and wheat flour. Target analytes included alternariol (AOH), AOH-3-glucoside, AOH-9-glucoside, AOH-3-sulfate, alternariol monomethyl ether (AME), AME-3-glucoside, AME-3-sulfate, altenuene, isoaltenuene, tenuazonic acid (TeA), tentoxin (TEN), altertoxin I and II, alterperylenol, stemphyltoxin III, altenusin, and altenuic acid III. Extensive optimization resulted in a time- and cost-effective sample preparation protocol and a chromatographic baseline separation of included isomers. Overall, adequate limits of detection (0.03–9 ng/g) and quantitation (0.6–18 ng/g), intermediate precision (9–44%), and relative recovery values (75–100%) were achieved. However, stemphyltoxin III, AOH-3-sulfate, AME-3-sulfate, altenusin, and altenuic acid III showed recoveries in wheat flour below 70%, while their performance was stable and reproducible. Our pilot study with samples from the Austrian retail market demonstrated that tomato sauces (n = 12) contained AOH, AME, TeA, and TEN in concentrations up to 20, 4, 322, and 0.6 ng/g, while sunflower seed oil (n = 7) and wheat flour samples (n = 9) were contaminated at comparatively lower levels. Interestingly and of relevance for risk assessment, AOH-9-glucoside, discovered for the first time in naturally contaminated food items, and AME-3-sulfate were found in concentrations similar to their parent toxins. In conclusion, the established multi-analyte method proved to be fit for purpose for generating comprehensive Alternaria toxin occurrence data in different food matrices. Graphical abstractᅟ Electronic supplementary material The online version of this article (10.1007/s00216-018-1105-8) contains supplementary material, which is available to authorized users.

The synthesis of 10 novel P-substituted dithienophosphole oxide compounds applying phenylcarbazole and indolocarbazole donors is presented. Based on photo-physical and theoretical investigations, the study reveals that the pyramidal geometry of the phosphorus allows for the synthesis of charge transfer materials by introducing strong exocyclic donor groups but suppresses intramolecular charge transfer below a certain donor strength threshold, which is an appealing structural feature for the design of donor–acceptor materials. The triplet energies of the phenylcarbazole based compounds are in the range of 2.49–2.65 eV, sufficiently high for potential applications as host materials in PhOLEDs. By contrast, the introduction of indolocarbazole, the weakest employed donor, yields materials exhibiting a significantly higher triplet energy of up to 2.87 eV and a remarkably low singlet–triplet splitting (0.18 eV). In addition an interesting example of an intramolecular electronic through-space interaction has been observed for the ortho-linked phenylcarbazole derivative.

Alternaria spp. are ubiquitous molds that are able to produce toxic secondary metabolites which may contaminate food globally. One of those is the mycotoxin altertoxin II (ATX-II), a genotoxic and mutagenic compound. In recent years, different flavonoids that may co-occur with mycotoxins in food were demonstrated to temper toxic effects of molds, mostly through their anti-oxidant properties. Thus, in this study, we assessed the influence of the berry anthocyanidin delphinidin on the toxicity of ATX-II in HT-29 colon carcinoma cells. We performed coupled SRB/WST-1 cytotoxicity assays which revealed only weak antagonistic interactions, and single-cell gel electrophoresis ("comet") assays, where we observed a potent protective effect of delphinidin on the DNA-damaging properties of ATX-II. Furthermore, we investigated the mechanism for this interaction. In the DCF assay delphinidin was found to reduce intracellular oxidative stress levels, which might contribute partly to the latter protection. However, LC-MS experiments showed that co-incubation of the mycotoxin with either delphinidin or its potential degradation product phloroglucinol aldehyde significantly decreased ATX-II concentrations in aqueous solutions, indicating that a direct chemical reaction of ATX-II with these components is likely responsible for the observed loss of toxicity. Our results indicate that delphinidin - and possibly other anthocyanins as well - might play a role in the protection of the gut from Alternaria-induced genotoxicity.

The trichothecene mycotoxin deoxynivalenol (DON) is a frequent food contaminant and has been associated with gastroenteritis and primary symptoms such as nausea, diarrhea, and vomiting in humans. DON is extensively metabolized in plants, animals, and humans resulting in metabolites of diverse chemical and toxicological properties. In humans glucuronidation and de-epoxydation but not sulfation have been described as routes of DON conjugation/detoxification. In fact, no analytical methodology for the detection of the latter in human samples was available so far. Here, we report the development of a highly sensitive LC-MS/MS method for the quantification of DON-sulfates together with other relevant DON metabolites in human urine for advanced bio-monitoring purpose. Authentic DON-sulfate standards were chemically synthetized and confirmed by NMR. For sample preparation a ‘dilute and shoot’ approach was chosen. Despite urine dilution convenient limits of detection below 0.6 ng/mL were achieved for DON-3-sulfate and DON-15-sulfate. The developed method was applied to a set of urine samples obtained from pregnant women who were exposed to DON through their normal diet. Many of the samples have been previously shown to contain DON in concentrations up to 275 ng/mL and even higher concentrations of DON- glucuronides. Using the developed method we were able to detect DON-3-sulfate in 29 out of 40 urine samples (72%). The maximum and median concentration of DON-3-sulfate was 56 ng/mL and 1.5 ng/mL, respectively contributing to about 3-4% of the total DON content in the tested samples (i.e. sum of DON, DON-3-glucuronide, DON-15-glucuronide, and DON-3-sulfate). Interestingly, no DON-15-sulfate was detected in any sample. Though the absolute concentrations of DON-3- sulfate were lower than those of DON- glucuronides, the detection of this novel metabolic pathway in humans opens up for new research questions. In fact, the contribution of sulfate metabolites to the overall toxicity of DON remains to be clarified. Therefore, in vitro experiments were pre this novel human excretion mechanism warrants more detailed investigation. Therefore, in vitro experiments were performed for a first toxicological characterization of DON-sulfates in human HT-29 colon cells. Several assays were carried out focusing on cellular activation of the Nrf2/ARE pathway. The overall results of these toxicological experiments suggested that DON-3- sulfate might not be considered as a human detoxification mechanism. In view of the first identification of this metabolite in human urine these results suggest a more detailed investigation of this newly discovered conjugate of the Fusarium toxin DON in human.