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Alternaria molds are known to cause the contamination of food with their secondary metabolites, a chemically very heterogeneous group of compounds. Yet, after decades of research on the occurrence and the toxicity of Alternaria toxins in academia, no regulation has been implemented yet, thus leaving these potential food contaminants in the status of so-called "emerging mycotoxins". However, research on this topic has been far from static, leading to the European Food Safety Authority repeatedly calling for more data on the occurrence and toxicity of genotoxic metabolites such as alternariol (AOH) and its monomethyl ether (AME). To give an overview on recent developments in the field, this comprehensive review summarizes published data and addresses current challenges arising from the chemical complexity of Alternaria's metabolome, mixture effects and the emergence of novel biological targets like cell membranes or the interaction with different receptors. Besides toxicodynamics, we review recent research on toxicokinetics, including the first in vivo studies which incorporated the rarely investigated-but highly genotoxic-perylene quinones. Furthermore, a particular focus lies on the advances of liquid chromatography/tandem mass spectrometry (LC-MS/MS)-based analytical tools for determining a broader spectrum of Alternaria toxins including modified/masked forms and assessing exposure via human biomonitoring (HBM).

Alternaria molds produce a variety of chemically diverse secondary metabolites with potentially adverse effects on human health. However, data on occurrence in food and human exposure is inconsistent for some of these mycotoxins. Membrane filtration is a frequent step in many sample preparation procedures for LC-MS-based methods analyzing food contaminants. Yet, little is known about the possibility of adsorptive phenomena that might result in analyte losses. Thus, we treated a complex extract of Alternaria toxins with several types of syringe filters and unraveled the impact on its chemical composition by LC-MS/MS. We observed significant, and in some cases complete, losses of compounds due to filtration. Particularly, two key Alternaria toxins, alternariol (AOH) and its monomethyl ether (AME), were heavily affected. As a comparison with published food surveys indicating a correlation of the type of filtration used with lower incidence reports in food, our results point at a possible underestimation of AME in past exposure assessment. Also, perylene quinones were greatly affected by filtration, underlining the importance to take this into consideration during analytical method development. Furthermore, we applied the comet assay in HT-29 cells to elucidate the impact of filtration on the genotoxicity of the extract. We observed strong coincidences with the loss of epoxide-carrying metabolites and also an intriguing induction of oxidative DNA damage by yet toxicologically uncharacterized Alternaria toxins. In conclusion, we highlight potential issues with sample filtration and call for a critical re-evaluation of previous food occurrence data in the light of the results at hand.

Exposure to food and environmental contaminants is a global environmental health issue. In this study, innovative LC-MS/MS approaches were applied to investigate mycotoxin co-exposure in mother-infant pairs (n = 23) by analyzing matched plate-ready food, breast milk and urine samples of mothers and their exclusively breastfed infants. The study revealed frequent co-occurrence of two to five mycotoxins. Regulated (e.g. aflatoxins, deoxynivalenol and ochratoxin A) and emerging mycotoxins (e.g. alternariol monomethyl ether and beauvericin) were frequently detected (3 %–89 % and 45 %–100 %), in at least one specimen. In addition, a moderate association of ochratoxin A in milk to urine of mothers (r = 0.47; p = 0.003) and infants (r = 0.52; p = 0.019) but no other significant correlations were found. Average concentration levels in food mostly did not exceed European maximum residue limits, and intake estimates demonstrated exposure below tolerable daily intake values. Infants were exposed to significantly lower toxin levels compared to their mothers, indicating the protective effect of breastfeeding. However, the transfer into milk and urine and the resulting chronic low-dose exposure warrant further monitoring. In the future, occurrence of mycotoxin-mixtures, and their combined toxicological effects need to be comprehensively considered and implemented in risk management strategies. These should aim to minimize early-life exposure in critical developmental stages.

Environmental exposure to xenoestrogens, i.e., chemicals that imitate the hormone 17β-estradiol, has the potential to influence hormone homeostasis and action. Detailed knowledge of xenobiotic biotransformation processes in cell models is key when transferring knowledge learned from in vitro models to in vivo relevance. This study elucidated the metabolism of two naturally-occurring phyto- and mycoestrogens; namely genistein and zearalenone, in an estrogen receptor positive breast cancer cell line (MCF-7) with the aid of stable isotope-assisted metabolomics and the bioinformatic tool MetExtract II. Metabolism was studied in a time course experiment after 2 h, 6 h and 24 h incubation. Twelve and six biotransformation products of zearalenone and genistein were detected, respectively, clearly demonstrating the abundant xenobiotic biotransformation capability of the cells. Zearalenone underwent extensive phase-I metabolism resulting in α-zearalenol (α-ZEL), a molecule known to possess a significantly higher estrogenicity, and several phase-II metabolites (sulfo- and glycoconjugates) of the native compound and the major phase I metabolite α-ZEL. Moreover, potential adducts of zearalenone with a vitamin and several hydroxylated metabolites were annotated. Genistein metabolism resulted in sulfation, combined sulfation and hydroxylation, acetylation, glucuronidation and unexpectedly adduct formation with pentose- and hexose sugars. Kinetics of metabolite formation and subsequent excretion into the extracellular medium revealed a time-dependent increase in most biotransformation products. The untargeted elucidation of biotransformation products formed during cell culture experiments enables an improved and more meaningful interpretation of toxicological assays and has the potential to identify unexpected or unknown metabolites.

The gut microbiome of neonates, infants, and toddlers (NITs) is very dynamic, and only begins to stabilize towards the third year of life. Within this period, exposure to xenobiotics may perturb the gut environment, thereby driving or contributing to microbial dysbiosis, which may negatively impact health into adulthood. Despite exposure of NITs globally, but especially in Africa, to copious amounts and types of xenobiotics - such as mycotoxins, pesticide residues, and heavy metals - little is known about their influence on the early-life microbiome or their effects on acute or long-term health. Within the African context, the influence of fermented foods, herbal mixtures, and the delivery environment on the early-life microbiome are often neglected, despite being potentially important factors that influence the microbiome. Consequently, data on in-depth understanding of the microbiome-exposome interactions is lacking in African cohorts. Collecting and evaluating such data is important because exposome-induced gut dysbiosis could potentially favor disease progression.

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.

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.

The human gut microbiota plays an important role in the maintenance of human health. Factors able to modify its composition might predispose the host to the development of pathologies. Among the various xenobiotics introduced through the diet, Alternaria mycotoxins are speculated to represent a threat for human health. However, limited data are currently available about the bidirectional relation between gut microbiota and Alternaria mycotoxins. In the present work, we investigated the in vitro effects of different concentrations of a complex extract of Alternaria mycotoxins (CE; containing eleven mycotoxins; e.g. 0.153 µM alternariol and 2.3 µM altersetin, at the maximum CE concentration tested) on human gut bacterial strains, as well as the ability of the latter to metabolize or adsorb these compounds. Results from the minimum inhibitory concentration assay showed the scarce ability of CE to inhibit the growth of the tested strains. However, the growth kinetics of most of the strains were negatively affected by exposure to the various CE concentrations, mainly at the highest dose (50 µg/mL). The CE was also found to antagonize the formation of biofilms, already at concentrations of 0.5 µg/mL. LC–MS/MS data analysis of the mycotoxin concentrations found in bacterial pellets and supernatants after 24 h incubation showed the ability of bacterial strains to adsorb some Alternaria mycotoxins, especially the key toxins alternariol, alternariol monomethyl ether, and altersetin. The tendency of these mycotoxins to accumulate within bacterial pellets, especially in those of Gram-negative strains, was found to be directly related to their lipophilicity.

BACKGROUND Infants are a vulnerable population whose nutrition changes as complementary foods are introduced; a process which may modify patterns of exposure to dietary mycotoxins. However, exposure monitoring of dietary mycotoxin mixtures in several biological samples obtained from breastfed and non-exclusively breastfed children is scarce. OBJECTIVES To examine mycotoxin co-exposure patterns in infants using a multi-specimen, multi-mycotoxin approach. METHODS Breast milk, complementary food and urine obtained from 65 infants, aged 1–18 months, in Ogun state, Nigeria, were analyzed for mycotoxins using ultra-sensitive LC-MS/MS approaches. RESULTS Complementary food was contaminated with seven distinct classes of mycotoxins including aflatoxins (9/42 samples; range: 1.0–16.2µg/kg) and fumonisins (14/42; range: 8– 167µg/kg). Aflatoxin M1 was detected in breast milk (4/22), while six other classes of mycotoxins were quantified; including dihydrocitrinone (6/22; range: 14.0–59.7ng/L) and sterigmatocystin (1/22; 1.2ng/L) detected for the first time. Mycotoxins were detected in 64/65 of the urine samples, with seven distinct classes of mycotoxins observed demonstrating ubiquitous exposure. Two aflatoxin metabolites (AFM1 and AFQ1) and FB1 were detected in 6/65, 44/65 and 17/65 samples, respectively. Mixtures of mycotoxin classes were common, including 14/42, 22/22 and 56/65 samples having 2–4, 2–6 or 2–6 mycotoxins present, for complementary food, breast milk and urine, respectively. Aflatoxin and/or fumonisin was detected in 12/14, 4/22 and 46/56 for complimentary foods, breast milk and urine, respectively. Furthermore, the detection frequency, mean concentrations and occurrence of mixtures were typically greater in urine of non-exclusively breastfed compared to breastfed children. CONCLUSIONS The study provides novel insights into mycotoxin co-exposures in children in a mycotoxin high-risk country without proper food safety measures. Albeit a small sample set, it highlights significant transition to higher levels of infant mycotoxin exposure as complementary foods are introduced, providing impetus to mitigate during this critical early-life period and encourage breastfeeding.

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.

In order to understand the changes in toxic metabolite profiles in uncooked and cooked foods, samples of flour/grain (n = 40) and their corresponding plate-ready food (n = 39) were collected from 40 households in two states of northern Nigeria. The food samples were analyzed for multiple fungal metabolites by LC-MS/MS and daily intakes of mycotoxins in the diets were estimated and compared to established margin of exposure (MOE) and tolerable daily intake (TDI) values. Both food groups contained 65 fungal and plant metabolites, inclusive of 23 mycotoxins. The mean concentrations of aflatoxin B1, beauvericin, fumonisin B1 (FB1), FB2, FB3, hydrolysed FB1, moniliformin and nivalenol were significantly (p < 0.05) higher in flour than in the plate-ready food samples. The levels of several mycotoxins were higher in the flour samples than in plate-ready meals by 129–383%. The dilution effect from proportionate mixing of flour samples with water led to 48–100% reduction in detectable mycotoxins in flour to plate-ready meals. Aflatoxins and fumonisins co- occurred in 36% of the plate-ready foods. Exposures of households to aflatoxins and fumonisins based on 95% CI concentration of mycotoxins in the meals were high, suggesting potential health risks based on calculated low MOE and exceedence of stipulated TDI value, respectively.

Human breast milk is considered as the best and ideal form of nutrition for infants. However, food contaminants such as mycotoxins, which may be transferred from maternal blood to milk, are poorly described. Mycotoxins are a major group of natural toxins frequently detected in foods. Here, we review the current state-of-the-art in the monitoring of mycotoxins in human breast milk, i.e., knowledge on occurrence, metabolism, and analytical assays utilized for their quantification. We highlight that most of the data captured to date have not been verified with the precision now capable utilizing LC-MS/MS and LC-HRMS approaches. One concern is that some studies may overestimate individual measures, and most cannot capture the patterns and levels of mycotoxin mixtures. We propose accurate assessment as a priority, especially for aflatoxins, fumonisins, ochratoxin A, zearalenone, and deoxynivalenol as well as their major metabolites. However, also so-called emerging toxins such as citrinin, the enniatins, beauvericin, aurofusarin, or Alternaria toxins should be considered to evaluate their potential relevance. Key requirements for analytical quality assurance are identified and discussed to guide future developments in this area. Moreover, research needs including investigations of lactational transfer rates, the role of human metabolism for bioactivation or detoxification, and an evaluation of potential combinatory effects of different mycotoxins are pointed out. It is hoped that LC-MS based multianalyte methods will enable more accurate, rapid and affordable human biomonitoring approaches that support informed decisions for maternal and infant health.

Exposure of humans and animals to mycotoxins via food and feed generally involves a conglomeration of compounds contaminating the consumed products. Investigations on combinatory effects of mycotoxins are therefore of great importance. In this study, cytotoxic effects of binary mixtures of the Fusarium toxins enniatin B, aurofusarin, deoxynivalenol, nivalenol and zearalenone, and tenuazonic acid produced by Alternaria spp., were evaluated by the WST-1 assay in the colorectal carcinoma cell-line Caco-2 after 24h of incubation. The selection of these mycotoxins was based on typically occurring natural contamination patterns in grains. Aurofusarin, which can be found abundantly in contaminated foodstuff and has not been toxicologically characterized properly so far, showed pronounced cytotoxicity, decreasing the mitochondrial activity at 10μM to 51% compared to a solvent control. Combinations of other mycotoxins with aurofusarin showed additive effects. In contrast, binary mixtures of enniatin B, deoxynivalenol, nivalenol and zearalenone at cytotoxic concentrations, predominantly resulted in antagonistic effects. Binary combinations of these four Fusarium toxins with tenuazonic acid also revealed interacting effects leading to a decrease in cytotoxicity, compared to expected combinatory effects. Especially in combination with deoxynivalenol, tenuazonic acid was found to significantly reduce the cytotoxicity of this mycotoxin in Caco-2 cells. Synergistic effects were not observed for any toxin combination under the chosen conditions.

There is a critical need to better understand the patterns, levels and combinatory effects of exposures we are facing through our diet and environment. Mycotoxin mixtures are of particular concern due to chronic low dose exposures caused by naturally contaminated food. To facilitate new insights into their role in chronic disease, mycotoxins and their metabolites are quantified in bio-fluids as biomarkers of exposure. Here, we describe a highly sensitive urinary assay based on ultra-high performance liquid chromatography - tandem mass spectrometer (UHPLC-MS/MS) and 13C-labelled or deuterated internal standards covering the most relevant regulated and emerging mycotoxins. Utilizing enzymatic pre-treatment, solid phase extraction and UHPLC separation, the sensitivity of the method was significantly higher (10-160x lower LODs) than in a previously described method used for comparison purpose, and stable isotopes provided compensation for challenging matrix effects. This method was in-house validated and applied to re-assess mycotoxin exposure in urine samples obtained from Nigerian children, adolescent and adults, naturally exposed through their regular diet. Owing to the methods high sensitivity, biomarkers were detected in all samples. The mycoestrogen zearalenone was the most frequently detected contaminant (82%) but also ochratoxin A (76%), aflatoxin M1 (73%) and fumonisin B1 (71%) were quantified in a large share of urines. Overall, 57% of 120 urines were contaminated with both, aflatoxin M1 and fumonisin B1, and other co-exposures were frequent. These results clearly demonstrate the advanced performance of the method to assess lowest background exposures (pg mL−1 range) using a single, highly robust assay that will allow for the systematic investigation of low dose effects on human health.

Infants are particularly susceptible toward the toxic effects of food contaminants, including mycotoxins. However, multimycotoxin exposure assessment in breast milk has received very limited attention so far, resulting in a poor understanding of coexposures during early life. Here, we present the development and application of a highly sensitive, specific, and quantitative assay assessing up to 28 mycotoxins, including regulated (aflatoxins, ochratoxin A, deoxynivalenol, zearalenone) and emerging mycotoxins as well as key metabolites by LC-MS/MS. After careful optimization of the sample preparation procedure, a QuEChERS protocol combined with a freeze-out step was validated in-house. The limits of quantification varied between 0.009 and 2.9 ng/mL, and for most analytes extraction recovery (74–116%) and intermediate precision (2–20%) were satisfactory. The method was applied to examine multiple breast milk samples obtained from 22 women (n = 75 in total) from Ogun State, Nigeria. Most samples were either entirely free of mycotoxins or contaminated to a minimal extent with beauvericin (56%), enniatin B (9%), ochratoxin A (15%), and aflatoxin M1 (1%). The most abundant mycotoxin was beauvericin, which was not reported in this biological fluid before, with concentrations up to 0.019 ng/mL. In conclusion, the method demonstrated to be fit for purpose to determine and quantify low background contaminations in human breast milk. On the basis of the high sensitivity of the novel analytical method, it was possible to deduce that tolerable daily intake values were not exceeded by breastfeeding in the examined infants.

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.

We report the identification of deoxynivalenol-3-sulfate and deoxynivalenol-15-sulfate as two novel metabolites of the trichothecene mycotoxin deoxynivalenol in wheat. Wheat ears which were either artificially infected with Fusarium graminearum or directly treated with the major Fusarium toxin deoxynivalenol (DON) were sampled 96 h after treatment. Reference standards, which have been chemically synthesized and confirmed by NMR, were used to establish a liquid chromatography-electrospray ionization (LC-ESI)-MS/MS-based “dilute and shoot” method for the detection, unambiguous identification, and quantification of both sulfate conjugates in wheat extracts. Using this approach, detection limits of 0.003 mg/kg for deoxynivalenol-3-sulfate and 0.002 mg/kg for deoxynivalenol-15-sulfate were achieved. Matrix-matched calibration was used for the quantification of DON-sulfates in the investigated samples. In DON-treated samples, DON-3-sulfate was detected in the range of 0.29–1.4 mg/kg fresh weight while DON-15-sulfate concentrations were significantly lower (range 0.015–0.061 mg/kg fresh weight). In Fusarium-infected wheat samples, DON-3-sulfate was the only detected sulfate conjugate (range 0.022–0.059 mg/kg fresh weight). These results clearly demonstrate the potential of wheat to form sulfate conjugates of DON. In order to test whether sulfation is a detoxification reaction in planta, we determined the ability of the sulfated DON derivatives to inhibit in vitro protein synthesis of wheat ribosomes. The results demonstrate that both DON-sulfates can be regarded as detoxification products. DON-15-sulfate was about 44× less inhibitory than the native toxin, and no toxicity was observed for DON-3-sulfate in the tested range. Graphical abstract ᅟ Electronic supplementary material The online version of this article (doi:10.1007/s00216-014-8340-4) contains supplementary material, which is available to authorized users.

Human exposures to mycotoxins through dietary intake are a major health hazard and may result in various pathophysiological effects. Although Thailand is a country at increased risk due to its climatic conditions, no comprehensive dataset is available to perform proper exposure assessment of its population with regard to mycotoxins. Therefore, this pilot study was conducted to investigate and evaluate the exposure levels of major mycotoxins (aflatoxin B₁, ochratoxin A, fumonisins, zearalenone and trichothecenes). Sixty first-morning urine samples were collected from healthy volunteers who live in the Bangkok metropolitan area and surrounding provinces (Pathumthani, Nonthaburi, Samutprakarn and Samutsakorn). Urine samples were analysed by a LC-MS/MS-based multi-biomarker method following a so-called 'dilute and shoot' approach. Results generally indicated low mycotoxin exposures in most individuals through the determination of the four biomarkers that were detected in urine samples, i.e. aflatoxin M₁, ochratoxin A (OTA), as well as the deoxynivalenol (DON) metabolites DON-3-glucuronide and DON-15-glucuronide in 10 of 60 individuals. The maximum concentrations were used to estimate the daily intake confirming that none of the individuals exceeded the tolerable daily intake (TDI) of DON (maximum 26% of TDI) or OTA (maximum 22% of TDI). However, the maximum exposure of aflatoxin B₁, estimated to be 0.91 µg (kg bw)⁻¹ day⁻¹, should raise some concerns and suggests further studies utilising a more sensitive method. Low exposure to Fusarium toxins was also confirmed by the absence of zearalenone, α-zearalanol, β-zearalanol and zearalenone-14-glucuronide as well as T-2 toxin, HT-2 toxin, nivalenol and free DON. This is the first multi-mycotoxin biomarker study performed in Southeast Asia.

The aim was to determine the mycotoxin transfer rate into beer during a semi-industrial production process and the effect of fungicide treatment in the field on mycotoxins concentrations in beer. To ensure the usual practical agronomical conditions, sample A was treated with fungicide Prosaro® 250, and sample B was infected with Fusarium culmorum spores, in order to obtain infected malt. Malt was produced using standard procedure and beer was produced in a semi-industrial unit. During fermentation measurement of sugars (maltotriose and maltose), glycerol and ethanol content was performed on a daily basis. Multiple toxins were determined in malt and beer. Deoxynivalenol (DON), its modified plant metabolite DON-3-glucoside (DON-glucoside), brevianamide F, tryptophol, linamarin, lotaustralin, culmorin (CUL), 15-hydroxy-CUL and 5-hydroyx-CUL were detected in all samples. Results indicate that F. culmorum infection did not influence the fermentation process or the alcohol concentration.

Xenoestrogenic mycotoxins may contaminate food and feed posing a public health issue. Besides the zearalenone group, the Alternaria toxin alternariol (AOH) has been described as a potential mycoestrogen. However, the estrogenicity of Alternaria toxins is still largely overlooked and further data are needed to better describe the group toxicity. In the frame of risk assessment, mixed in silico/in vitro approaches already proved to be effective first-line analytical tools. An integrated in silico/in vitro approach was used to investigate the effects of metabolic and chemical modifications on the estrogenicity of AOH. Among the considered modifications, methylation was found critical for enhancing estrogenicity (as seen for alternariol monomethyl ether (AME)) while hydroxylation and glucuronidation had the opposite effect (as seen for 4-hydroxy AOH and 4-hydroxy AME). The structure-activity relationship analysis provided the structural rationale. Our results provide insights to design more efficient risk assessment studies expanding knowledge over the group toxicity.

Toxins of microbial origin frequently contaminate foodstuffs worldwide and pose a serious hazard to humans. This study reports on LCMS/MS quantification of multiple fungal and bacterial toxins, from household sampling of 50 traditionally prepared maize-fufu samples from Bamunka village, western highlands of Cameroon. Seventy-four metabolites including aflatoxin B1 (AFB1) (12/50: mean 0.9, range n.d-1.8 μg kg −1 ), cereulide (50/50: mean 37; range 1–236 μg kg −1 ), deoxynivalenol (DON) (50/50: mean 23, range 14–55 μg kg −1 ), fumonisin B1 (FB1) (50/50: mean: 151, range 48–709 μg kg −1 ), nivalenol (NIV) (50/50; mean 268, range 116–372 μg kg −1 ), patulin (PAT) (15/50:mean 105, range 12–890 μg kg −1 ) and zearalenone (ZEN) (50/50: mean 49, range 5–150) were detected; and of note every sample contained at least 27 toxic compounds. While individual toxin levels were mostly low there is always concern regarding mixtures, for which data are absent or limited. This study reports several novel observations of toxins not previously reported in maize, and the mixture of toxins, e.g. cereulide, PAT and ZEN derivatives (ZEN-cis and ZENsulfate-cis) are reported for the first time in Cameroonian food.

The microbiological safety of spontaneously fermented foods is not always guaranteed due to the undefined fermenting microbial consortium and processing materials. In this study, two commonly consumed traditional condiments (iru and ogiri) and their respective raw seeds (locust bean and melon) purchased from markets in south-western Nigeria were assessed for bacterial diversity and mycotoxin contamination using 16S rRNA gene sequencing and liquid chromatography tandem mass spectrometry (LC-MS/MS), respectively. Two hundred isolates obtained from the raw seeds and condiments clustered into 10 operational taxonomic units (OTUs) and spanned 3 phyla, 10 genera, 14 species and 2 sub-species. Bacillus (25%) and Staphylococcus (23.5%) dominated other genera. Potentially pathogenic species such as Alcaligenes faecalis, Bacillus anthracis, Proteus mirabilis and Staphylococcus sciuri subsp. sciuri occurred in the samples, suggesting poor hygienic practice during production and/or handling of the condiments. A total of 48 microbial metabolites including 7 mycotoxins [3-nitropropionic acid, aflatoxin B1 (AFB1), AFB2, beauvericin, citrinin, ochratoxin A and sterigmatocystin] were quantified in the food samples. Melon and ogiri had detectable aflatoxin levels whereas locust bean and iru did not; the overall mycotoxin levels in the food samples were low. There is a need to educate processors/vendors of these condiments on good hygienic and processing practices.

A pilot, cross-sectional, correlational study was conducted in eight rural communities in northern Nigeria to investigate mycotoxin exposures in 120 volunteers (19 children, 20 adolescents and 81 adults) using a modern LC–MS/MS based multi-biomarker approach. First morning urine samples were analyzed and urinary biomarker levels correlated with mycotoxin levels in foods consumed the day before urine collection. A total of eight analytes were detected in 61/120 (50.8%) of studied urine samples, with ochratoxin A, aflatoxin M1 and fumonisin B1 being the most frequently occurring biomarkers of exposure. These mycotoxin biomarkers were present in samples from all age categories, suggestive of chronic (lifetime) exposures. Rough estimates of mycotoxin intake suggested some exposures were higher than the tolerable daily intake. Overall, rural consumer populations from Nasarawa were more exposed to several mixtures of mycotoxins in their diets relative to those from Kaduna as shown by food and urine biomarker data. This study has shown that mycotoxin co-exposure may be a major public health challenge in rural Nigeria; this calls for urgent intervention. Keywords: Biomarker, Exposure assessment, Food safety, Human urine, Mycotoxin biomonitoring, Nigeria

Seventy composite samples of maize grains stored in five agro-ecological zones (AEZs) of Nigeria where maize is predominantly produced were evaluated for the presence of microbial metabolites with the LC-MS/MS technique. The possible relationships between the storage structures and levels of mycotoxin contamination were also evaluated. Sixty-two fungal and four bacterial metabolites were extracted from the grains, 54 of which have not been documented for maize in Nigeria. Aflatoxin B1 and fumonisin B1 were quantified in 67.1 and 92.9 % of the grains, while 64.1 and 57.1 % exceeded the European Union Commission maximum acceptable limit (MAL) for aflatoxin B1 and fumonisins, respectively. The concentration of deoxynivalenol was, however, below the MAL with occurrence levels of 100 and 10 % for its masked metabolite, deoxynivalenol glucoside. The bacterial metabolites had low concentrations and were not a source of concern. The storage structures significantly correlated positively or negatively (p

Subsistence farmers are exposed to a range of mycotoxins. This study applied novel urinary multi-mycotoxin LC-MS/MS methods to determine multiple exposure biomarkers in the high oesophageal cancer region, Transkei, South Africa. Fifty-three female participants donated part of their maize-based evening meal and first void morning urine, which was analysed both with sample clean-up (single and multi-biomarker) and by a 'dilute-and-shoot' multi-biomarker method. Results were corrected for recovery with LOD for not detected. A single biomarker method detected fumonisin B1 (FB1) (87% incidence; mean ± standard deviation 0.342 ± 0.466 ng/mg creatinine) and deoxynivalenol (100%; mean 20.4 ± 49.4 ng/mg creatinine) after hydrolysis with b-glucuronidase. The multi-biomarker 'dilute-and-shoot' method indicated deoxynivalenol-15-glucuronide was predominantly present. A multi-biomarker method with b-glucuronidase and immunoaffinity clean-up determined zearalenone (100%; 0.529 ± 1.60 ng/mg creatinine), FB1 (96%; 1.52 ± 2.17 ng/mg creatinine), a-zearalenol (92%; 0.614 ± 1.91 ng/mg creatinine), deoxynivalenol (87%; 11.3 ± 27.1 ng/mg creatinine), b-zearalenol (75%; 0.702 ± 2.95 ng/mg creatinine) and ochratoxin A (98%; 0.041 ± 0.086 ng/mg creatinine). These demonstrate the value of multi-biomarker methods in measuring exposures in populations exposed to multiple mycotoxins. This is the first finding of urinary deoxynivalenol, zearalenone, their conjugates, ochratoxin A and zearalenols in Transkei.

In this pilot survey the levels of various mycotoxin biomarkers were determined in third trimester pregnant women from eastern Croatia. First void urine samples were collected and analysed using a "dilute and shoot" LC-ESI-MS/MS multi biomarker method. Deoxynivalenol (DON) and its metabolites: deoxynivalenol-15-glucuronide and deoxynivalenol-3-glucuronide were detected in 97.5% of the studied samples, partly at exceptionally high levels, while ochratoxin A was found in 10% of the samples. DON exposure was primarily reflected by the presence of deoxynivalenol-15-glucuronide with a mean concentration of 120 μg L(-1), while free DON was detected with a mean concentration of 18.3 μg L(-1). Several highly contaminated urine samples contained a third DON conjugate, tentatively identified as deoxynivalenol-7-glucuronide by MS/MS scans. The levels of urinary DON and its metabolites measured in this study are the highest ever reported, and 48% of subjects were estimated to exceed the provisional maximum tolerable daily intake (1 μg kg(-1) b.w.).

Maize harvested in the Centane region of the former Transkei, Eastern Cape Province, South Africa, by subsistence farmers has been shown over many seasons to be contaminated with fumonisin mycotoxins. However, there are limited data on the presence of other mycotoxins. Two multimycotoxin LC-MS/MS methods were applied to good and moldy maize samples, as separated by the farmers themselves from the 2011 harvest. One method involved extract cleanup on multitoxin immunoaffinity columns before LC-MS/MS analysis for aflatoxins, fumonisins, deoxynivalenol (DON), zearalenone (ZEN), and T-2 and HT-2 toxins. The other method was based on a "dilute-and-shoot" approach for the above mycotoxins and a wide range of other fungal secondary metabolites. Both methods showed high incidences of fumonisins B1 and B2 (FB1 and FB2) in good maize (100% for both by the first method, means were 2083 and 927 μg/kg for the two analogues; 93% for both by the second method, positive means of 2764 and 1050 μg/kg, respectively). All samples of moldy maize were contaminated (mean FB1 of 27.64 and 35.98 mg/kg, respectively; mean FB2 of 10.58 and 14.14 mg/kg, respectively). Comparison of the two methods for FB1 and FB2 over the entire range of samples gave R(2) values 0.9144 and 0.8859, respectively. Low levels of DON were found by both methods (positive means of 12 and 4.7 μg/kg in good maize, respectively, and of 14 and 5.8 μg/kg in moldy maize, respectively). ZEN was determined with positive means of 108 and 25 μg/kg in good maize, respectively, and of 111 and 135 μg/kg in moldy maize, respectively. No aflatoxins, OTA, or T-2 or HT-2 toxins were detected. A wide range of other Fusarium , Aspergillus , Alternaria , and Penicillium mycotoxins and secondary metabolites were determined.

Mycotoxins are toxic fungal secondary metabolites that frequently contaminate food and feed worldwide, and hence represent a major hazard for food and feed safety. To estimate human exposure arising from contaminated food, so-called biomarker approaches have been developed as a complementary biomonitoring tool besides traditional food analysis. The first methods based on radioimmunoassays and enzyme-linked immunosorbent assays as well as on liquid chromatography were developed in the late 1980s and early 1990s for the carcinogenic aflatoxins and in the last two decades further tailor-made methods for some major mycotoxins have been published. Since 2010, there has been a clear trend towards the development and application of multianalyte methods based on liquid chromatography–electrospray ionization tandem mass spectrometry for assessment of mycotoxin exposure made possible by the increased sensitivity and selectivity of modern mass spectrometry instrumentation and sophisticated sample cleanup approaches. With use of these advanced methods, traces of mycotoxins and relevant breakdown and conjugation products can be quantified simultaneously in human urine as so-called biomarkers and can be used to precisely describe the real exposure, toxicokinetics, and bioavailability of the toxins present. In this article, a short overview and comparison of published multibiomarker methods focusing on the determination of mycotoxins and relevant excretion products in human urine is presented. Special attention is paid to the main challenges when analyzing these toxic food contaminants in urine, i.e., very low analyte concentrations, appropriate sample preparation, matrix effects, and a lack of authentic, NMR-confirmed calibrants and reference materials. Finally, the progress in human exposure assessment studies facilitated by these analytical methods is described and an outlook on probable developments and possibilities is presented. Figure Mycotoxin exposure assessment: traditional food analysis compared to the innovative, complementary biomarker approach

The fungal and multi-mycotoxin profiles of groundnuts sold in domestic markets in Nigeria as well as the associated risk to consumers were assessed in the present study. Four hundred fungal isolates representing mainly Aspergillus [58.6%: Aspergillus section Flavi (37.1%) and A. niger- clade (21.5%)], Penicillium (40.9%) and Fusarium (0.5%) were isolated from 82 (97.6%, n = 84) groundnut samples collected from four agro-ecological zones (AEZs) of Nigeria. The incidence of aflatoxin-producing A. flavus isolates (71%) was significantly ( p 1 , AFB 2 , AFG 1 , AFG 2 and AFM 1 ), beauvericin (BEAU), cyclopiazonic acid (CPA), moniliformin, nivalenol and ochratoxin A] and four bacterial metabolites were detected in the groundnuts by liquid chromatography tandem mass spectrometry. Aflatoxins (39%; max: 2076 μg/kg; mean: 216 μg/kg) were detected in more samples than any other mycotoxin. About 25, 23 and 14% of the samples respectively were above the 2 μg/kg AFB 1 , 4 and 20 μg/kg total aflatoxin limits of the European Union and US FDA respectively. The mean margins of exposure of AFB 1 and total aflatoxins for adult consumers were 1665 and 908, respectively, while mean estimated daily intake values for infants, children and adults were

A preliminary survey involving limited sample size was conducted to determine the spectrum of moulds and mycotoxins in wheat grains from flour mills and local markets in Nigeria. Fourteen wheat samples were analyzed for moulds using standard mycological methods and for toxic fungal metabolites using a liquid chromatography-tandem mass spectrometric method. Fusarium (range of incidence 12.5–61.7%) dominated in the wheat grains though species of Aspergillus (range of incidence 2.24–3.86%) were also recovered from the samples. The identified fungal species were Aspergillus flavus (7.7%), Aspergillus niger clade (2.6%), Fusarium avenaceum (10.9%), Fusarium culmorum (22.4%) and Fusarium graminearum (56.4%). A total of 54 microbial metabolites were detected in the samples at concentration ranging between 0.01 μg/kg for macrosporin and 2560 μg/kg for deoxynivalenol. Among the four mycotoxins addressed by regulations in the European Union (EU) found in the samples, deoxynivalenol (incidence 100%) dominated in the samples and its levels exceeded the maximum acceptable EU limit (750 μg/kg) in 36% of the samples. This report underscores the need for more robust surveys with larger sample sizes and across several agro-ecologies in the country.

The Fusarium toxin deoxynivalenol (DON) is one of the most abundant mycotoxins worldwide and poses many adverse health effects to human and animals. Consequently, regulatory limits and a provisional maximum tolerable daily intake (PMTDI) for this important type B-trichothecene were assigned. We conducted a pilot survey to investigate the level of DON exposure in Austrian adults by measurements of DON and its glucuronide conjugates (DON-GlcA's), as biomarkers of exposure, in first morning urine. The average concentration of total DON (free DON+DON-GlcA's) was estimated to be 20.4±2.4 μg L⁻¹ (max. 63 μg L⁻¹). Surprisingly, we found that one third of the volunteers (n=27) exceeded the established PMTDI when consuming regular diet. DON-GlcA's were directly quantified by LC-MS/MS and the results were compared with indirect quantification after enzymatic hydrolysis and confirmed the suitability of the direct method. Moreover, we investigated the in vivo metabolism of DON in humans and were able to determine two closely eluting DON-GlcA's in naturally contaminated urine samples for the first time. In contrast to previous findings we have tentatively identified DON-15-glucuronide as a major DON metabolite in human urine based on the analysis of these samples. About 75% of total glucuronides were derived from this metabolite while DON-3-glucuronide accounted for approximately 25%. The reported new findings clearly demonstrate the great potential of suitable biomarkers to critically assess exposure of humans and animals to DON.

Nucleotide sugars, the activated forms of monosaccharides, are important metabolites involved in a multitude of cellular processes including glycosylation of xenobiotics. Especially in plants, UDP-glucose is one of the most prominent members among these nucleotide-sugars, as it is involved in the formation of glucose conjugates of xenobiotics, including mycotoxins, but also holds a central role in the interconversion of energized sugars such as the formation of UDP-glucuronic acid required for cell wall biosynthesis. Here, we present the first HILIC-LC-ESI-TQ-MS/MS method for the quantification of UDP-glucose and UDP-glucuronic acid together with the Fusarium toxin deoxynivalenol (DON) and its major plant detoxification product DON-3-O-glucoside (DON-3-Glc) utilizing a polymer-based column. For sample preparation a time-effective and straightforward 'dilute and shoot' protocol was applied. The chromatographic run time was minimized to 9min including proper column re-equilibration. In-house validation of the method verified its linear range, intra- (1-7%) and interday (8-20%) precision, instrumental LODs between 0.6 and 10ngmL(-1), selectivity and moderate matrix effects with mean recoveries of 85-103%. To prove the methods applicability, we analyzed two sets of wheat extracts obtained from different cultivars grown under standardized greenhouse conditions. The results clearly demonstrated the suitability of the developed method to quantify UDP-glucose, DON and its masked form D3G in diluted wheat extracts. We observed differing concentration levels of UDP-glucose in the two wheat cultivars showing different resistance to the severe plant disease Fusarium head blight. We propose that the higher ability to detoxify DON into DON-3-Glc might be a consequence of the higher cellular UDP-glucose pool in the resistant cultivar.

Deoxynivalenol (DON) is one of the most abundant mycotoxins contaminating food and feed worldwide. Upon absorption, the major portion of the toxin is excreted by humans and animal species as glucuronide. However, consistent in vitro data on DON glucuronidation are lacking. In the present study, the metabolism of DON was investigated using liver microsomes from humans and six different animal species. It was shown that all animal and human liver microsomes led to the formation of up to three different mono-O-glucuronides with significant interspecies differences. While the activity of human liver microsomes was low (0.8 to 2.2 pmol·min(-1)·mg(-1)), bovine liver and rat liver microsomes conjugated DON with activities of 525 pmol·min(-1)·mg(-1) and 80 pmol·min(-1)·mg(-1), respectively.

For the first time in Cameroon, this paper reports on multiple mycotoxins occurrences in poultry feeds. Twenty feed samples collected from different poultry farms were analyzed for 320 fungal metabolites by liquid chromatography electrospray ionization tandem mass spectrometry. Results showed feeds contamination by 68 metabolites including 18 mycotoxins/metabolites currently regulated in the European Union such as fumonisins B1 (FB1), B2, and B3; deoxynevalenol (DON); and beta-zearalenol recovered in all samples. FB1 reported highest FB mean level of 468 (range 16-1930) microg kg(-1). Levels of DON and ZEN were mostly concentrated in feeds from western-highlands conversely for FBs and aflatoxins concentrations in Yaounde. Aflatoxin B1 mean level of 40 microg kg(-1) exceeded the worldwide permitted limit for aflatoxins in feed and generally inversely proportional to weight gain in chicken.

Metabolites of toxigenic fungi and bacteria occur as natural contaminants (e.g. mycotoxins) in feedstuffs making them unsafe to animals. The multi-toxin profiles in 58 commercial poultry feed samples collected from 19 districts in 17 states of Nigeria were determined by LC/ESI-MS/MS with a single extraction step and no clean-up. Sixty-three (56 fungal and seven bacterial) metabolites were detected with concentrations ranging up to 10,200 µg kg⁻¹ in the case of aurofusarin. Fusarium toxins were the most prevalent group of fungal metabolites, whereas valinomycin occurred in more than 50% of the samples. Twelve non-regulatory fungal and seven bacterial metabolites detected and quantified in this study have never been reported previously in naturally contaminated stored grains or finished feed. Among the regulatory toxins in poultry feed, aflatoxin concentrations in 62% of samples were above 20 µg kg⁻¹, demonstrating high prevalence of unsafe levels of aflatoxins in Nigeria. Deoxynivalenol concentrations exceeded 1000 µg kg⁻¹ in 10.3% of samples. Actions are required to reduce the consequences from regulatory mycotoxins and understand the risks of the single or co-occurrence of non-regulatory metabolites for the benefit of the poultry industry.

Mycotoxins regularly occur in food worldwide and pose serious health risks to consumers. Since individuals can be exposed to a variety of these toxic secondary metabolites of fungi at the same time, there is a demand for proper analytical methods to assess human exposure by suitable biomarkers.This study reports on the development of a liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) method for the quantitative measurement of 15 mycotoxins and key metabolites in human urine using polarity switching. Deoxynivalenol (DON), DON-3-O-glucuronide, DON-15-O-glucuronide (D15GlcA), de-epoxy DON, nivalenol (NIV), T-2 toxin, HT-2 toxin, zearalenone, zearalenone-14-O-glucuronide, α- and β-zearalenol, fumonisins B(1) and B(2) (FB(1), FB(2)), ochratoxin A (OTA) and aflatoxin M(1) (AFM(1)) were determined without the need for any cleanup using a rapid and simple dilute and shoot approach.Validation was performed in the range of 0.005-40 µg L(-1) depending on the analyte and expected urinary concentration levels. Apparent recoveries between 78 and 119% and interday precisions of 2-17% relative standard deviation (RSD) were achieved. The applicability of the method was demonstrated by the analysis of urine samples obtained from Cameroon. In naturally contaminated urine samples up to six biomarkers of exposure (AFM(1), DON, D15GlcA, NIV, FB(1), and OTA) were detected simultaneously.We conclude that the developed LC/MS/MS method is well suited to quantify multiple mycotoxin biomarkers in human urine down to the sub-ppb range within 18 min and without any prior cleanup. The co-occurrence of several mycotoxins in the investigated samples clearly emphasizes the great potential and importance of this method to assess exposure of humans and animals to naturally occurring mycotoxins.