Geographic tracing of cereals from South Tyrol (Italy) and neighboring regions via 87Sr/86Sr isotope analysis.

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 87Sr/86Sr ratios. Soil and grain (different species) samples were collected from around 100 fields in two sampling campaigns. No difference in the 87Sr/86Sr ratios among different cereal species (p < 0.05) was found when cultivated on the same field. A high correlation between 87Sr/86Sr ratios in cereal grains and soil samples was found, with results in line with the local geology characteristics. Cereal samples from South Tyrol showed relatively high 87Sr/86Sr values (0.716 - 0.721, mean 50%), separating them from the other regions investigated and many cereal production areas of global importance.

Markers for DNA damage are induced in the rat colon by the Alternaria toxin altertoxin-II, but not a complex extract of cultured Alternaria alternata.

Mycotoxins produced by Alternaria spp. act genotoxic in cell-based studies, but data on their toxicity in vivo is scarce and urgently required for risk assessment. Thus, male Sprague-Dawley rats received single doses of a complex Alternaria toxin extract (CE; 50 mg/kg bw), altertoxin II (ATX-II; 0.21 mg/kg bw) or vehicle by gavage, one of the most genotoxic metabolites in vitro and were sacrificed after 3 or 24 h, respectively. Using SDS-PAGE/Western Blot, a significant increase of histone 2a.X phosphorylation and depletion of the native protein was observed for rats that were exposed to ATX-II for 24 h. Applying RT-PCR array technology we identified genes of interest for qRT-PCR testing, which in turn confirmed an induction of Rnf8 transcription in the colon of rats treated with ATX-II for 3 h and CE for 24 h. A decrease of Cdkn1a transcription was observed in rats exposed to ATX-II for 24 h, possibly indicating tissue repair after chemical injury. In contrast to the observed response in the colon, no markers for genotoxicity were induced in the liver of treated animals. We hereby provide the first report of ATX-II as a genotoxicant in vivo. Deviating results for similar concentrations of ATX-II in a natural Alternaria toxin mixture argue for substantial mixture effects.

Gut microbiota and undigested food constituents modify toxin composition and suppress the genotoxicity of a naturally occurring mixture of Alternaria toxins in vitro.

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.

Bioavailability, metabolism, and excretion of a complex Alternaria culture extract versus altertoxin II: a comparative study in rats.

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, < 0.06% in urine and plasma, < 5% in feces) or not at all (highly genotoxic, epoxide-holding altertoxin II, stemphyltoxin III). Interestingly, altertoxin I was detected in all matrices of rats receiving altertoxin II and suggests enzymatic de-epoxidation in vivo. In conclusion, the present study contributes valuable information to advance our understanding of the emerging Alternaria mycotoxins and their relevance on food safety.

Naturally occurring mixtures of Alternaria toxins: anti-estrogenic and genotoxic effects in vitro.

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 17ß-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.

The Fate of Altertoxin II During Tomato Processing Steps at a Laboratory Scale.

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 <1% after 24 h. In heated purees (100°C for 30 min after ATX-II addition), also only minor concentrations accounting for 2-4% were determined. Moreover, the reduction of the compound's epoxide group to the alcohol, i.e., the formation of altertoxin I was demonstrated in intact tomato fruits (7-12%), suggesting enzymatic biotransformation of the xenobiotic by the plant's metabolism.

First insights into Alternaria multi-toxin in vivo metabolism.

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.

Response of intestinal HT-29 cells to the trichothecene mycotoxin deoxynivalenol and its sulfated conjugates.

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.

Tracking emerging mycotoxins in food: development of an LC-MS/MS method for free and modified Alternaria toxins.

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 abstract ᅟ.

Delphinidin protects colon carcinoma cells against the genotoxic effects of the mycotoxin altertoxin II.

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 secondary Fusarium metabolite aurofusarin induces oxidative stress, cytotoxicity and genotoxicity in human colon cells.

Aurofusarin (AURO), a dimeric naphthoquinone, is produced by Fusarium fungi. Although frequently found in food and feed, toxicological studies are limited. Hence, the in vitro toxicity of AURO was investigated in the colon adenocarcinoma cell line HT29 and the non-tumorigenic colon cells HCEC-1CT. Cytotoxic effects were found at concentrations ≥1 µM by evaluating mitochondrial activity (WST-1) and cellular proliferation (sulforhodamine B assay). 10 µM of AURO induced a decrease of cells in the S-phase, measured by flow cytometry. Confocal microscopy revealed AURO-mediated increase of intracellular p53 protein. In accordance, DNA-damage was seen in the comet assay (≥1 µM) together with enhanced levels of formamidopyrimidine-DNA-glycosylase (fpg)-sensitive sites, indicative for oxidative stress. An increase of intracellular reactive oxygen species was observed in the dichlorofluorescein (DCF) assay (≥5 µM). The GSSG/GSH ratio was elevated, but no impact on redox-sensitive Nrf2-dependent genes (Nrf2, γ-GCL, NQO1) was found at the gene expression level. However, induction of cytochrome P450 monooxygenase (CYP) 1A1 was measured at the gene expression and protein level. In conclusion, these in vitro data suggest that, when co-occurring, AURO might be considered as a potential contributor to the overall toxicity of respective Fusarium mycotoxin mixtures.

Identification of a novel human deoxynivalenol metabolite enhancing proliferation of intestinal and urinary bladder cells.

The mycotoxin deoxynivalenol (DON) is an abundant contaminant of cereal based food and a severe issue for global food safety. We report the discovery of DON-3-sulfate as a novel human metabolite and potential new biomarker of DON exposure. The conjugate was detectable in 70% of urine samples obtained from pregnant women in Croatia. For the measurement of urinary metabolites, a highly sensitive and selective LC-MS/MS method was developed and validated. The method was also used to investigate samples from a duplicate diet survey for studying the toxicokinetics of DON-3-sulfate. To get a preliminary insight into the biological relevance of the newly discovered DON-sulfates, in vitroexperiments were performed. In contrast to DON, sulfate conjugates lacked potency to suppress protein translation. However, surprisingly we found that DON-sulfates enhanced proliferation of human HT-29 colon carcinoma cells, primary human colon epithelial cells (HCEC-1CT) and, to some extent, also T24 bladder cancer cells. A proliferative stimulus, especially in tumorigenic cells raises concern on the potential impact of DON-sulfates on consumer health. Thus, a further characterization of their toxicological relevance should be of high priority.