Search

Early human life is considered a critical window of susceptibility to external exposures. Infants are exposed to a multitude of environmental factors, collectively referred to as the exposome. The chemical exposome can be summarized as the sum of all xenobiotics that humans are exposed to throughout a lifetime. We review different exposure classes and routes that impact fetal and infant metabolism and the potential toxicological role of mixture effects. We also discuss the progress in human biomonitoring and present possible models for studying maternal-fetal transfer. Data gaps on prenatal and infant exposure to xenobiotic mixtures are identified and include natural biotoxins, in addition to commonly reported synthetic toxicants, to obtain a more holistic assessment of the chemical exposome. We highlight the lack of large-scale studies covering a broad range of xenobiotics. Several recommendations to advance our understanding of the early-life chemical exposome and the subsequent impact on health outcomes are proposed. Expected final online publication date for the

Polyphenols are investigated intensively in nutritional and clinical settings because of their beneficial bioactive properties and prevalence in plant-based foods. Due to their complexity, analysis with untargeted approaches is favorable. These approaches typically use high-resolution mass spectrometry (HRMS) rather than low-resolution mass spectrom-etry (LRMS). The advantages of using HRMS were evaluated in this work by thoroughly testing various untargeted techniques and available online resources. By applying iterative data-dependent acquisition on real-life urine samples, 27 features were identified with spectral libraries, 88 with in silico fragmentation, and 113 features were annotated by their monoisotopic mass with PhytoHub, an online database containing >2000 polyphenols. Moreover, potentially polyphenol-related features were searched for using various non-targeted analysis techniques including MassQL for glucuronide and sulfate neutral losses, and MetaboAnalyst for statistical analysis. As HRMS typically suffers from a sensitivity loss compared to state-of-the-art LRMS used in targeted workflows, the sensitivity gap between the two instrumental approaches was quantified. Both instruments showed feasible sensitivity, with the median limits of detection being 10 - 18 ng/mL for HRMS and 4.8 - 5.8 ng/mL for LRMS in three human matrices (urine, serum, and plas-ma). The applicability of HRMS was further evaluated in real-life urine samples with 24 polyphenols detected. The results demonstrate that despite its intrinsic limitations, HRMS can readily be used for comprehensively investigating human exposure and related health effects of polyphenols and their metabolites as well as toxicological mixture effects with other xenobiotics.

Data on the natural occurrence of Alternaria mycotoxins in apple by-products is lacking in Argentina and the risk of exposure to these mycotoxins has not been characterized before. The levels of alternariol (AOH), alternariol monomethyl ether (AME), altenuene (ALT), tenuazonic acid (TeA), tentoxin (TEN), altertoxin-I (ATX-I), altertoxin-II (ATX-II), alternariol 3-sulfate (AOH-3-S), alternariol 3-glucoside (AOH-3-G), alternariol monomethyl ether 3-sulfate (AME-3-S), and alternariol monomethyl ether 3-glucoside (AME-3-G) were determined in clarified and cloudy apple juices, marmalades, and apple-based infant food from the Argentinean market, and the risk of exposure was characterized. Detectable levels of AME, TEN, TeA, AME-3-S and AOH-3-G were found in clarified juices, while the same mycotoxins plus AOH were found in cloudy apple juices in higher concentrations. AME, TEN, TeA and AOH-3G were detected in marmalades, and AOH, AME, TEN and TeA in apple infant food. Probabilistic exposure assessment and risk characterization were carried out for children between 6 months and 5 years old in Argentina. The highest risk of exposure affected children between 6 and 23 months from the consumption of apple infant food and mainly associated with the alternariols. Better control strategies to prevent the incorporation of Alternaria mouldy core into the process line and the establishment of legislation for Alternaria mycotoxins are needed in Argentina.

Apple fruits are susceptible to infection with Alternaria species, mainly causing mouldy core disease, hindering the detection of infected fruit by processing industries. Data on the natural occurrence of Alternaria mycotoxins in apple by-products is lacking in Argentina. The risk of exposure to these mycotoxins has not been characterized before. Therefore, the levels of alternariol (AOH), alternariol monomethyl ether (AME), altenuene (ALT), tenuazonic acid (TeA), tentoxin (TEN), altertoxin-I (ATX-I), altertoxin-II (ATX-II), alternariol 3-sulfate (AOH-3-S), alternariol 3-glucoside (AOH-3-G), alternariol monomethyl ether 3-sulfate (AME-3-S), and alternariol monomethyl ether 3-glucoside (AME-3-G) were determined in clarified and cloudy apple juices, marmalades, and apple-based infant food from the Argentinean market, and the risk of exposure was characterized. In the clarified juices, detectable levels of AME, TEN, TeA, AME-3-S and AOH-3-G were found, while in the cloudy apple juices the same mycotoxins plus AOH were found in higher concentrations. AME, TEN, TeA and AOH-3G were found in marmalades, and AOH, AME, TEN and TeA were detected in apple infant food. Probabilistic exposure assessment and risk characterization were carried out for children between 6 months and 5 years old in Argentina from the consumption of clarified and cloudy apple juices and apple-based infant food. The highest risk of exposure affected children between 6 and 23 months from the consumption of apple infant food. The alternariols represented the highest risk. Better control strategies to prevent the incorporation of Alternaria mouldy core into the process line, and the establishment of legislation for Alternaria mycotoxins are needed in Argentina.

The totality of environmental exposures and lifestyle factors, commonly referred to as the exposome, is poorly understood. Measuring the myriad of chemicals that humans are exposed to is immensely challenging, and identifying disrupted metabolic pathways is even more complex. Here, we present a novel technological approach for the comprehensive, rapid, and integrated analysis of the endogenous human metabolome and the chemical exposome. By combining reverse-phase and hydrophilic interaction liquid chromatography (HILIC) and fast polarity-switching, molecules with highly diverse chemical structures can be analyzed in 15 min with a single analytical run as both column's effluents are combined before analysis. Standard reference materials and authentic standards were evaluated to critically benchmark performance. Highly sensitive median limits of detection (LODs) with 0.04 μM for140 quantitatively assessed endogenous metabolites and 0.08 ng/mL for the100 model xenobiotics and human estrogens in solvent were obtained. In matrix, the median LOD values were higher with 0.7 ng/mL (urine) and 0.5 ng/mL (plasma) for exogenous chemicals. To prove the dual-column approach's applicability, real-life urine samples from sub-Saharan Africa (high-exposure scenario) and Europe (low-exposure scenario) were assessed in a targeted and nontargeted manner. Our liquid chromatography high-resolution mass spectrometry (LC-HRMS) approach demonstrates the feasibility of quantitatively and simultaneously assessing the endogenous metabolome and the chemical exposome for the high-throughput measurement of environmental drivers of diseases.

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).

The chemical exposome consists of all environmental exposures experienced throughout a lifetime. Liquid chromatography high-resolution mass spectrometry (HRMS) is common in untargeted exposome-wide analyses of xenobiotics in biological samples; however, human biomonitoring approaches usually utilize targeted low-resolution triple quadrupole (QQQ) mass spectrometry tailored to a small number of chemicals. HRMS can identify novel contaminants over a broad mass range but the detection of molecules from low-level exposure amidst a background of highly-abundant endogenous molecules has proven to be difficult. In this study, high- and low-resolution mass spectrometry with identical chromatography was utilized to determine the limits of quantitation (LOQ) of >100 xenobiotics and estrogenic hormones in pure solvent and human urine. For HRMS analyses, the median LOQ was 0.9 and 1.2 ng/mL in solvent and urine, respectively and for low-resolution QQQ measurements, the median LOQ was 0.1 and 0.2 ng/mL in solvent and urine, respectively. To evaluate the calculated LOQs in complex biological samples, spot urine samples from 24 Nigerian females were measured. The higher LOQ values for HRMS resulted in less quantified low-abundance analytes and decreased the number of compounds detected below the LOQ. Even at chronic low-dose exposure, such compounds might be relevant for human health because of a high individual toxicity or potential mixture effects. Nevertheless, HRMS enabled the additional screening for exposure to unexpected/unknown analytes, including emerging compounds and biotransformation products. Therefore, a synergy between high- and low-resolution mass spectrometry may currently be the best option to elucidate and quantify xenobiotics in exposome-wide association studies (ExWAS).

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.

Humans are exposed to many xenobiotics simultaneously, but little is known about the toxic effects based on chemical-chemical interactions. This study aims at evaluating the binary interactions between 13 common environmental organic compounds (resulting in 78 pairs) by observing their cytotoxicity on HepG2 cells. Among all of the tested pairs, the combination of flame-retardant triphenyl phosphate (TPP) and tris(1,3-dichloro-2-propyl)phosphate (TDCPP) exhibited one of the most significant synergistic effects. We further characterized the transcriptome and metabolome after combined exposure to TPP and TDCPP and individual exposure. The results suggested that the coexposure caused many more changes in gene expressions and cellular activities. The transcriptome data showed that the coexposure triggered significant pathway changes including "cholesterol biosynthesis" and "ATF6-Alpha activated chaperone genes", together with distinct gene ontology (GO) terms such as the "negative regulation of the ERK1 and ERK2 cascade". Additionally, coexposure enhanced the biological activity of liver X receptors and nuclear factor erythroid 2-related factor 2 (Nrf2). The metabolome data showed that coexposure significantly elevated oxidative stress and affected the purine and pyrimidine metabolism. Overall, this study showed that interactions, which may enhance or suppress the biological processes, are common among environmental chemicals, although their environmental relevance should be studied in the future.

African American (AA) smokers are at a higher risk of developing lung cancer compared to whites. The variations in the metabolism of nicotine and tobacco-derived carcinogens in these groups were reported previously with the levels of nicotine metabolites and carcinogen-derived metabolites measured using targeted approaches. While useful, these targeted strategies are not able to detect global metabolic changes for use in predicting the detrimental effects of tobacco use and ultimately lung cancer susceptibility among smokers. To address this limitation, we have performed global untargeted metabolomics profiling in urine of AA and white smokers to characterize the pattern of metabolites, identify differentially regulated pathways, and correlate these profiles with the observed variations in lung cancer risk between these two populations. Urine samples from AA (n = 30) and white (n = 30) smokers were used for metabolomics analysis acquired in both positive and negative electrospray ionization modes. LC-MS data were uploaded onto the cloud-based XCMS online (http://xcmsonline.scripps.edu) platform for retention time correction, alignment, feature detection, annotation, statistical analysis, data visualization, and automated systems biology pathway analysis. The latter identified global differences in the metabolic pathways in the two groups including the metabolism of carbohydrates, amino acids, nucleotides, fatty acids, and nicotine. Significant differences in the nicotine degradation pathway (cotinine glucuronidation) in the two groups were observed and confirmed using a targeted LC-MS/MS approach. These results are consistent with previous studies demonstrating AA smokers with lower glucuronidation capacity compared to whites. Furthermore, the d-glucuronate degradation pathway was found to be significantly different between the two populations, with lower amounts of the putative metabolites detected in AA compared to whites. We hypothesize that the differential regulation of the d-glucuronate degradation pathway is a consequence of the variations in the glucuronidation capacity observed in the two groups. Other pathways including the metabolism of amino acids, nucleic acids, and fatty acids were also identified, however, the biological relevance and implications of these differences across ethnic groups need further investigation. Overall, the applied metabolomics approach revealed global differences in the metabolic networks and endogenous metabolites in AA and whites, which could be used and validated as a new potential panel of biomarkers that could be used to predict lung cancer susceptibility among smokers in population-based studies.

Xenobiotics are ubiquitous in the environment and modified in the human body by phase I and II metabolism. Liquid chromatography coupled to high resolution mass spectrometry is a powerful tool to investigate these biotransformation products. We present a workflow based on stable isotope-assisted metabolomics and the bioinformatics tool MetExtract II for deciphering xenobiotic metabolites produced by human cells. Its potential was demonstrated by the investigation of the metabolism of deoxynivalenol (DON), an abundant food contaminant, in a liver carcinoma cell line (HepG2) and a model for colon carcinoma (HT29). Detected known metabolites included DON-3-sulfate, DON-10-sulfonate 2, and DON-10-glutathione as well as DON-cysteine. Conjugation with amino acids and an antibiotic was confirmed for the first time. The approach allows the untargeted elucidation of human xenobiotic products in tissue culture. It may be applied to other fields of research including drug metabolism, personalized medicine, exposome research, and systems biology to better understand the relevance of in vitro experiments.

Xenobiotics are chemicals foreign to a specific organism that humans are exposed to on a daily basis through their food, drugs and the environment. These molecules are frequently metabolized to increase polarity and subsequent excretion. During sample preparation, deconjugation of phase II metabolites is a critical step to capture the total exposure to chemicals in liquid chromatography mass spectrometry assays (LC-MS). Knowledge on deconjugation efficiencies of different enzymes and the extend of conjugation in human biofluids has primarily been investigated for single compounds or individual chemical classes. In this study, the performance of three β-glucuronidase and arylsulfatase mixtures from H. pomatia, from recombinant sources (BGS™), and from Escherichia coli combined with recombinant arylsulfatase (ASPC™) was compared and the efficiency of phase II deconjugation was monitored in breast milk, urine and plasma. An innovative LC-MS/MS biomonitoring method encompassing more than 80 highly diverse xenobiotics (e.g., plasticizers, industrial chemicals, mycotoxins, phytoestrogens, pesticides) was utilized for the comprehensive investigation of phase II conjugation in experiments investigating levels in breast milk and urine obtained from breastfeeding women. Overall, it was confirmed that H. pomatia is the most efficient enzyme in hydrolyzing different classes of xenobiotics for future exposome-scale biomonitoring studies. The recombinant BGS™ formulation, however, provided better results for breast milk samples, primarily due to lower background contamination, a major issue when employing the typically applied crude H. pomatia extracts. A deeper understanding of the global xenobiotic conjugation patterns will be essential for capturing environmental and food-related exposures within the exposome framework more comprehensively.Graphical abstract

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.

Non-targeted analysis (NTA) encompasses a rapidly evolving set of mass spectrometry techniques aimed at characterizing the chemical composition of complex samples, identifying unknown compounds, and/or classifying samples, without prior knowledge regarding the chemical content of the samples. Recent advances in NTA are the result of improved and more accessible instrumentation for data generation and analysis tools for data evaluation and interpretation. As researchers continue to develop NTA approaches in various scientific fields, there is a growing need to identify, disseminate, and adopt community-wide method reporting guidelines. In 2018, NTA researchers formed the Benchmarking and Publications for Non-Targeted Analysis Working Group (BP4NTA) to address this need. Consisting of participants from around the world and representing fields ranging from environmental science and food chemistry to 'omics and toxicology, BP4NTA provides resources addressing a variety of challenges associated with NTA. Thus far, BP4NTA group members have aimed to establish a consensus on NTA-related terms and concepts and to create consistency in reporting practices by providing resources on a public Web site, including consensus definitions, reference content, and lists of available tools. Moving forward, BP4NTA will provide a setting for NTA researchers to continue discussing emerging challenges and contribute to additional harmonization efforts.

Non-targeted analysis (NTA) workflows using mass spectrometry are gaining popularity in many disciplines, but universally accepted reporting standards are nonexistent. Current guidance addresses limited elements of NTA reporting-most notably, identification confidence-and is insufficient to ensure scientific transparency and reproducibility given the complexity of these methods. This lack of reporting standards hinders researchers' development of thorough study protocols and reviewers' ability to efficiently assess grant and manuscript submissions. To overcome these challenges, we developed the NTA Study Reporting Tool (SRT), an easy-to-use, interdisciplinary framework for comprehensive NTA methods and results reporting. Eleven NTA practitioners reviewed eight published articles covering environmental, food, and health-based exposomic applications with the SRT. Overall, our analysis demonstrated that the SRT provides a valid structure to guide study design and manuscript writing, as well as to evaluate NTA reporting quality. Scores self-assigned by authors fell within the range of peer-reviewer scores, indicating that SRT use for self-evaluation will strengthen reporting practices. The results also highlighted NTA reporting areas that need immediate improvement, such as analytical sequence and quality assurance/quality control information. Although scores intentionally do not correspond to data/results quality, widespread implementation of the SRT could improve study design and standardize reporting practices, ultimately leading to broader use and acceptance of NTA data.

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.

Compartmentalization of the gut microbiota is thought to be important to system function, but the extent of spatial organization in the gut ecosystem remains poorly understood. Here, we profile the murine colonic microbiota along longitudinal and lateral axes using laser capture microdissection. We found fine-scale spatial structuring of the microbiota marked by gradients in composition and diversity along the length of the colon. Privation of fiber reduces the diversity of the microbiota and disrupts longitudinal and lateral gradients in microbiota composition. Both mucus-adjacent and luminal communities are influenced by the absence of dietary fiber, with the loss of a characteristic distal colon microbiota and a reduction in the mucosa-adjacent community, concomitant with depletion of the mucus layer. These results indicate that diet has not only global but also local effects on the composition of the gut microbiota, which may affect function and resilience differently depending on location., Here, employing laser capture microdissection and omics, the authors determine the effects of fiber-deprived diet on the spatial structure of the murine colon microbiome, finding that the absence of dietary fiber and polysaccharides leads to local changes along the colon and deterioration of the mucus layer.

We are constantly exposed to a variety of environmental contaminants and hormones, including those mimicking endogenous estrogens. These highly heterogeneous molecules are collectively referred to as xenoestrogens and hold the potential to affect and alter the delicate hormonal balance of the human body. To monitor exposure and investigate potential health implications, comprehensive analytical methods covering all major xenoestrogen classes are needed but not available to date. Herein, we describe a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous determination of multiple classes of endogenous as well as exogenous estrogens in human urine, serum, and breast milk to enable proper exposure and risk assessment. In total, 75 analytes were included, whereof a majority was successfully in-house validated in the three matrices. Extraction recoveries of validated analytes ranged from 71% to 110% and limits of quantification from 0.015 to 5 μg/L, 0.03 to 14 μg/L, and 0.03 to 4.6 μg/L in urine, serum, and breast milk, respectively. The applicability of the novel method was demonstrated in proof-of-principle experiments by analyzing urine from Austrian individuals and breast milk from Austrian and Nigerian individuals. Thereby, we proved the methods' feasibility to identify and quantify different classes of xenoestrogens simultaneously. The results illustrate the general importance of multiclass exposure assessment in the context of the exposome paradigm. Specifically, they highlight the need for estimating total estrogenic burden rather than single analyte or chemical class measurements and its potential impact in endocrine disruption and hormone related diseases including cancers.

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.

Establishing a method for human biomonitoring (HBM) of polyphenols enables the assessment of internal concentrations of these food bio-actives and the correlation with potential health effects such as antioxidant or anti-inflammatory properties. Thus, a targeted LC-MS/MS method for quantifying up to 90 analytes, representing the main polyphenol classes including flavanones, isoflavones, stilbenes, and phenolic acids, was developed for human urine, serum, and plasma. The method was established for low sample volumes and with a cost and time efficient sample preparation protocol for high-throughput, which is critical for its application in large cohort and exposome-wide association studies. On average, the sample preparation yielded extraction efficiencies of 98% for urine, 98% for serum, and 87% for plasma. Limits of detection were between 0.11 ng mL

DON, NX-3 and butenolide (BUT) are secondary metabolites formed by Fusarium graminearum. Evidence for formation of DON-glutathione adducts exists in plants, and also in human liver (HepG2) cells mass spectrometric evidence for GSH-adduct formation was reported. NX-3 is a DON derivative lacking structural features for Thiol-Michael addition, while BUT has the structural requirements (conjugated double bond and keto group). In the present study, we addressed whether these structural differences affect levels of intracellular reactive oxygen species in HepG2 cells, and if intracellular GSH levels influence toxic effects induced by DON, NX-3 and BUT. Pre-treatment with an inhibitor of GSH bio-synthesis, L-buthionine-[S,R]-sulfoximine, aggravated substantially BUT-induced cytotoxicity (≥50 μM, 24 h), but only marginally affected the cytotoxicity of DON and NX-3 indicating that GSH-mediated detoxification is of minor importance in HepG2 cells. We further investigated whether BUT, a compound inducing alone low oral toxicity, might affect the toxicity of DON. Under different experimental designs with respect to pre- and/or co-incubations, BUT was found to contribute to the combinatorial cytotoxicity, exceeding the toxic effect of DON alone. The observed combinatorial effects underline the potential contribution of secondary metabolites like BUT, considered to be alone of low toxicological relevance, to the toxicity of DON or structurally related trichothecenes, arguing for further studies on the toxicological relevance of naturally occurring mixtures.