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Within the EuroMix project, we have previously developed an adverse outcome pathway (AOP)-based in vitro assay toolbox to investigate the combined effects of liver steatosis-inducing compounds in human HepaRG hepatocarcinoma cells. In this study, we applied the toolbox to further investigate mixture effects of combinations, featuring either similarly acting or dissimilarly acting substances. The valproic acid structural analogs 2-propylheptanoic acid (PHP) and 2-propylhexanoic acid (PHX) were chosen for establishing mixtures of similarly acting substances, while a combination with the pesticidal active substance clothianidin (CTD) was chosen for establishing mixtures of dissimilarly acting compounds. We first determined relative potency factors (RPFs) for each compound based on triglyceride accumulation results. Thereafter, equipotent mixtures were tested for nuclear receptor activation in transfected HepG2 cells, while gene expression and triglyceride accumulation were investigated in HepaRG cells, following the proposed AOP for liver steatosis. Dose addition was observed for all combinations and endpoints tested, indicating the validity of the additivity assumption also in the case of the tested mixtures of dissimilarly acting substances. Gene expression results indicate that the existing steatosis AOP can still be refined with respect to the early key event (KE) of gene expression, in order to reflect the diversity of molecular mechanisms underlying the adverse outcome. Supplementary Information The online version contains supplementary material available at 10.1007/s00204-021-03182-1.

Since the addition of fluoride to drinking water in the 1940s, there have been frequent and sometimes heated discussions regarding its benefits and risks. In a recently published review, we addressed the question if current exposure levels in Europe represent a risk to human health. This review was discussed in an editorial asking why we did not calculate benchmark doses (BMD) of fluoride neurotoxicity for humans. Here, we address the question, why it is problematic to calculate BMDs based on the currently available data. Briefly, the conclusions of the available studies are not homogeneous, reporting negative as well as positive results; moreover, the positive studies lack control of confounding factors such as the influence of well-known neurotoxicants. We also discuss the limitations of several further epidemiological studies that did not meet the inclusion criteria of our review. Finally, it is important to not only focus on epidemiological studies. Rather, risk analysis should consider all available data, including epidemiological, animal, as well as in vitro studies. Despite remaining uncertainties, the totality of evidence does not support the notion that fluoride should be considered a human developmental neurotoxicant at current exposure levels in European countries.

Co-occurrence of pesticide residues in food commodities raises a potential safety issue as their mixture effects on human health are largely unknown. In a previous study, we reported the toxicological effects (pathology and histopathology) of imazalil (IMZ), thiacloprid (THI), and clothianidin (CTD) alone and in binary mixtures in a 28-day oral gavage study in female Wistar rats. Five dose levels (up to 350 mg/kg body weight/day) ranging from a typical toxicological reference value to a clear effect dose were applied. In the present study, we undertook a transcriptomics analysis of rat livers by means of total RNA sequencing (RNA-Seq). Bioinformatic data analysis involving Ingenuity Pathway Analysis (IPA) was used to gain mechanistic information on hepatotoxicity-related pathways affected after treatment with the pesticides, alone and in mixtures. Our data show that 2986 genes were differentially regulated by CTD while IMZ and THI had effects on 194 and 225 genes, respectively. All three individual compounds shared a common subset of genes whose network is associated with xenobiotic metabolism and nuclear receptor activation. Similar networks were retrieved for the mixtures. Alterations in the expression of individual genes were in line with the assumption of dose addition. Our results bring new insight into the hepatotoxicity mechanisms of IMZ, THI, and CTD and their mixtures. Supplementary Information The online version contains supplementary material available at 10.1007/s00204-020-02969-y.

Fatty acid esters of 3-chloropropane-1, 2-diol (3-MCPD) are food processing contaminants that are widespread in refined vegetable oils and fats, as well as in vegetable fat–containing products including infant formulas. The presence of these compounds in foods is of concern because of possible dangers for human health. Data from the recent in vivo (toxicokinetic) studies in rats shows that a substantial percentage of 3-MCPD could be released from 3-MCPD esters by enzymatic hydrolysis in the gastrointestinal tract and distributed to blood, organs, and tissues. The liberation of 3-MCPD from the esters is of toxicological concern, since 3-MCPD is known to induce tumors in long-term studies on rodents. The kidney and the male reproductive system have been identified as the main target organs for 3-MCPD-induced toxicity. The toxicity of the 3-MCPD esters is primarily associated with their potential to release the parent compound during digestion in the gastrointestinal tract. The recent in vivo data indicate that 3-MCPD esters could by hydrolyzed in the gastrointestinal tract and thus should be considered a potential source of human exposure to 3-MCPD. This chapter discusses the toxicology of 3-MCPD and toxicological properties of the esters. It concludes that the toxicological data for 2-MCPD esters is insufficient to conduct any accurate risk assessment at the moment.

Background: Exposure of consumers to aluminum-containing nanomaterials (Al NMs) through numerous products is an area of concern for public health agencies since human health risks are not completely elucidated. In addition, the available data on the genotoxicity of Al2O3 and Al0 NMs are inconclusive or rare. In order to provide further information, the present study investigated the in vitro genotoxic potential of Al0 and Al2O3 NMs in intestinal and liver cell models since these tissues represent organs which would be in direct contact or could experience potential accumulation following oral exposure. Methods: Differentiated human intestinal Caco-2 and hepatic HepaRG cells were exposed to Al0 and Al2O3 NMs (0.03 to 80 µg/cm2) and the results were compared with those obtained with the ionic form AlCl3. Several methods, including H2AX labelling, the alkaline comet assay and micronucleus (MN) assays were used. Oxidative stress and oxidative DNA damage were assessed using High Content Analysis (HCA) and the formamidopyrimidine DNA-glycosylase -modified comet assay respectively. Moreover, carcinogenic properties of Al NMs were investigated through the cell transforming assay (CTA) in Bhas 42 cells.Results: The three forms of Al did not induce chromosomal damage when tested in the MN assay. Furthermore, no cell transformation was observed in Bhas 42 cells. However, although no production of oxidative stress was detected in HCA assays, Al2O3 NMs induced oxidative DNA damage in Caco-2 cells in the comet assay following a 24 h treatment. Considerable DNA damage was observed with Al0 NMs in both cell lines in the comet assay, although this was likely due to interference with these NMs. Finally, no genotoxic effects were observed with AlCl3. Conclusion: The slight effects observed with Al NMs are therefore not likely to be related to ion release in the cell media.

Piperine is a natural ingredient of Piper nigrum (black pepper) and some other Piper species. Compared to the use of pepper for food seasoning, piperine is used in food supplements in an isolated, concentrated form and ingested as a bolus. The present review focuses on the assessment of the possible critical health effects regarding the use of isolated piperine as a single ingredient in food supplements. In human and animal studies with single or short-term bolus application of isolated piperine, interactions with several drugs, in most cases resulting in increased drug bioavailability, were observed. Depending on the drug and extent of the interaction, such interactions may carry the risk of unintended deleteriously increased or adverse drug effects. Animal studies with higher daily piperine bolus doses than in human interaction studies provide indications of disturbance of spermatogenesis and of maternal reproductive and embryotoxic effects. Although the available human studies rarely reported effects that were regarded as being adverse, their suitability for detailed risk assessment is limited due to an insufficient focus on safety parameters apart from drug interactions, as well as due to the lack of investigation of the potentially adverse effects observed in animal studies and/or combined administration of piperine with other substances. Taken together, it appears advisable to consider the potential health risks related to intake of isolated piperine in bolus form, e.g., when using certain food supplements.

ZnO nanoparticles are found in different food and consumer products, and their toxicological effects are still under investigation. It is therefore important to understand their behavior in the gas...

Pyrrolizidine alkaloids (PAs) are widely occurring phytotoxins which can induce severe liver damage in humans and other mammalian species by mechanisms that are not fully understood. Therefore, we investigated the development of PA hepatotoxicity in vivo, using an acutely toxic dose of the PA senecionine in mice, in combination with intravital two-photon microscopy, histology, clinical chemistry, and in vitro experiments with primary mouse hepatocytes and liver sinusoidal endothelial cells (LSECs). We observed pericentral LSEC necrosis together with elevated sinusoidal marker proteins in the serum of senecionine-treated mice and increased sinusoidal platelet aggregation in the damaged tissue regions. In vitro experiments showed no cytotoxicity to freshly isolated LSECs up to 500 µM senecionine. However, metabolic activation of senecionine by preincubation with primary mouse hepatocytes increased the cytotoxicity to cultivated LSECs with an EC50 of approximately 22 µM. The cytochrome P450 (CYP)-dependency of senecionine bioactivation was confirmed in CYP reductase-deficient mice where no PA-induced hepatotoxicity was observed. Therefore, toxic metabolites of senecionine are generated by hepatic CYPs, and may be partially released from hepatocytes leading to destruction of LSECs in the pericentral region of the liver lobules. Analysis of hepatic bile salt transport by intravital two-photon imaging revealed a delayed uptake of a fluorescent bile salt analogue from the hepatic sinusoids into hepatocytes and delayed elimination. This was accompanied by transcriptional deregulation of hepatic bile salt transporters like Abcb11 or Abcc1. In conclusion, senecionine destroys LSECs although the toxic metabolite is formed in a CYP-dependent manner in the adjacent pericentral hepatocytes.

Okadaic acid (OA) is a lipophilic phycotoxin that accumulates in the hepatopancreas and fatty tissue of shellfish. Consumption of highly OA-contaminated seafood leads to diarrhetic shellfish poisoning which provokes severe gastrointestinal symptoms associated with a disruption of the intestinal epithelium. Since the molecular mechanisms leading to intestinal barrier disruption are not fully elucidated, we investigated the influence of OA on intestinal tight junction proteins (TJPs) in differentiated Caco-2 cells. We found a concentration- and time-dependent deregulation of genes encoding for intestinal TJPs of the claudin family, occludin, as well as zonula occludens (ZO) 1 and 2. Immunofluorescence staining showed concentration-dependent effects on the structural organization of TJPs already after treatment with a subtoxic but human-relevant concentration of OA. In addition, changes in the structural organization of cytoskeletal F-actin as well as its associated protein ZO-1 were observed. In summary, we demonstrated effects of OA on TJPs in intestinal Caco-2 cells. TJP expressions were affected after treatment with food-relevant OA concentrations. These results might explain the high potential of OA to disrupt the intestinal barrier in vivo as its first target. Thereby the present data contribute to a better understanding of the OA-dependent induction of molecular effects within the intestinal epithelium.

Pyrrolizidine alkaloids (PA) are widely distributed phytotoxins contaminating food and feed. Hepatic enzymes are considered to bioactivate PA. Previous studies showed differences in the metabolism rate in liver homogenates of different species. Thus, uncertainty remains with respect to the relevance of human metabolism. Our study aimed to analyze whether the PA representative lasiocarpine is toxified by human cytochrome P450 (CYP) enzymes. We compared the metabolic elimination of lasiocarpine in the presence of rat and human S9 fractions and liver microsomes. Experiments with the potent CYP3A/Cyp3a inhibitor ketoconazole and supersomes containing individual human and rat CYPs revealed that enzymes of the CYP3A/Cyp3a family of both species are of major relevance for lasiocarpine metabolism. To assess if metabolism by human CYP3A4 results in a toxification of lasiocarpine we performed experiments with V79 cells. γH2AX and micronucleus formation were analyzed as endpoints for genotoxicity. No effects were observed in the wildtype cells, which lack CYP activity. By contrast, a V79 clone engineered for expression of human CYP3A4 showed concentration-dependent γH2AX and micronucleus formation. Concluding, our results showed the CYP3A4-dependent formation of genotoxic metabolites of lasiocarpine. The results confirm previous data indicating the need to include metabolism of PA for human risk assessment.

Drug-induced liver injury (DILI) cannot be accurately predicted by animal models. In addition, currently available in vitro methods do not allow for the estimation of hepatotoxic doses or the determination of an acceptable daily intake (ADI). To overcome this limitation, an in vitro/in silico method was established that predicts the risk of human DILI in relation to oral doses and blood concentrations. This method can be used to estimate DILI risk if the maximal blood concentration (Cmax) of the test compound is known. Moreover, an ADI can be estimated even for compounds without information on blood concentrations. To systematically optimize the in vitro system, two novel test performance metrics were introduced, the toxicity separation index (TSI) which quantifies how well a test differentiates between hepatotoxic and non-hepatotoxic compounds, and the toxicity estimation index (TEI) which measures how well hepatotoxic blood concentrations in vivo can be estimated. In vitro test performance was optimized for a training set of 28 compounds, based on TSI and TEI, demonstrating that (1) concentrations where cytotoxicity first becomes evident in vitro (EC10) yielded better metrics than higher toxicity thresholds (EC50); (2) compound incubation for 48 h was better than 24 h, with no further improvement of TSI after 7 days incubation; (3) metrics were moderately improved by adding gene expression to the test battery; (4) evaluation of harmacokinetic parameters demonstrated that total blood compound concentrations and the 95%-population-based percentile of Cmax were best suited to estimate human toxicity. With a support vector machine-based classifier, using EC10 and Cmax as variables, the cross-validated sensitivity, specificity and accuracy for hepatotoxicity prediction were 100, 88 and 93%, respectively. Concentrations in the culture medium allowed extrapolation to blood concentrations in vivo that are associated with a specific probability of hepatotoxicity and the corresponding oral doses were obtained by reverse modeling. Application of this in vitro/in silico method to the rat hepatotoxicant pulegone resulted in an ADI that was similar to values previously established based on animal experiments. In conclusion, the proposed method links oral doses and blood concentrations of test compounds to the probability of hepatotoxicity.

Evidence exists that humans are exposed to plastic microparticles via diet. Data on intestinal particle uptake and health-related effects resulting from microplastic exposure are scarce. Aim of the study was to analyze the uptake and effects of microplastic particles in human in vitro systems and in rodents in vivo. The gastrointestinal uptake of microplastics was studied in vitro using the human intestinal epithelial cell line Caco-2 and thereof-derived co-cultures mimicking intestinal M-cells and goblet cells. Different sizes of spherical fluorescent polystyrene (PS) particles (1, 4 and 10 µm) were used to study particle uptake and transport. A 28-days in vivo feeding study was conducted to analyze transport at the intestinal epithelium and oxidative stress response as a potential consequence of microplastic exposure. Male reporter gene mice were treated three times per week by oral gavage with a mixture of 1 µm (4.55 × 107 particles), 4 µm (4.55 × 107 particles) and 10 µm (1.49 × 106 particles) microplastics at a volume of 10 mL/kg/bw. Effects of particles on macrophage polarization were investigated using the human cell line THP-1 to detect a possible impact on intestinal immune cells. Altogether, the results of the study demonstrate the cellular uptake of a minor fraction of particles. In vivo data show the absence of histologically detectable lesions and inflammatory responses. The particles did not interfere with the differentiation and activation of the human macrophage model. The present results suggest that oral exposure to PS microplastic particles under the chosen experimental conditions does not pose relevant acute health risks to mammals.

Triazoles are commonly used fungicides which show liver toxicity in rodent studies. While hepatocellular hypertrophy is the most prominent finding, some triazoles have also been reported to cause hepatocellular steatosis. The aim of our study was to elucidate molecular mechanisms of triazole-mediated steatosis. Therefore, we used the two triazoles propiconazole (Pi) and tebuconazole (Te) as test compounds in in vitro assays using the human hepatocarcinoma cell lines HepG2 and HepaRG. Triglyceride accumulation was measured using the Adipored assay and by a gas-chromatographic method. Reporter gene analyses were used to assess the ability of Pi and Te to activate nuclear receptors, which are described as the molecular initiators in the adverse outcome pathway (AOP) for liver steatosis. The expression of steatosis-associated genes was investigated by RT-PCR. Mechanistic analyses of triazole-mediated steatosis were performed using HepaRG subclones that are deficient in different nuclear receptors. Pi and Te both interacted with the constitutive androstane receptor (CAR), the peroxisome proliferator-activated receptor alpha (PPARα), and the pregnane X receptor (PXR). Both compounds induced expression of steatosis-related genes and cellular triglyceride accumulation. The knockout of PXR in HepaRG cells, but not the CAR knockout, abolished triazole-induced triglyceride accumulation, thus underlining the crucial role of PXR in hepatic steatosis resulting from exposure to these fungicides. In conclusion, our findings provide new insight into the molecular mechanisms of steatosis induction by triazole fungicides and identify PXR as a critical mediator of this process.

International audience; Aluminum (Al) can be ingested from food and released from packaging and can reach key organs involved in human metabolism, including the liver via systemic distribution. Recent studies discuss the occurrence of chemically distinct Al-species and their interconversion by contact with biological fluids. These Al species can vary with regard to their intestinal uptake, systemic transport, and therefore could have species-specific effects on different organs and tissues. This work aims to assess the in vitro hepatotoxic hazard potential of three different relevant Al species: soluble AlCl3 and two nanoparticulate Al species were applied, representing for the first time an investigation of metallic nanoparticles besides to mineral bound gamma-Al2O3 on hepatic cell lines. To investigate the uptake and toxicological properties of the Al species, we used two different human hepatic cell lines: HepG2 and differentiated HepaRG cells. Cellular uptake was determined by different methods including light microscopy, transmission electron microscopy, side-scatter analysis, and elemental analysis. Oxidative stress, mitochondrial dysfunction, cell death mechanisms, and DNA damage were monitored as cellular parameters. While cellular uptake into hepatic cell lines occurred predominantly in the particle form, only ionic AlCl3 caused cellular effects. Since it is known, that Al species can convert one into another, and mechanisms including 'trojan-horse'-like uptake can lead to an Al accumulation in the cells. This could result in the slow release of Al ions, for which reason further hazard cannot be excluded. Therefore, individual investigation of the different Al species is necessary to assess the toxicological potential of Al particles.

BACKGROUND Case reports suggest a link between energy drinks (EDs) and adverse events, including deaths. OBJECTIVES We examined cardiovascular and metabolic effects of EDs and mixtures providing relevant ingredients of EDs compared to a similarly composed control product (CP) without these components. METHODS This randomized, crossover trial comprised 38 adults (19 women, mean BMI 23 kg/m2, mean age 22 y). We examined effects of a single administration of a commercial ED, the CP, and the CP supplemented with major ED-ingredients at the same concentrations as in the ED. The study products were administered at 2 volumes, 750 or 1000 mL. RESULTS Both volumes of the study products were acceptably tolerated with no dose-dependent effects on blood pressure (BP, primary outcome), heart rate, heart rate corrected duration of QT-segment in electrocardiography (QTc interval), and glucose metabolism. After ED consumption, 11% of the participants reported symptoms, in contrast to 0-3% caused by other study products. After 1 h, administration of an ED caused an increase in systolic BP (116.9 ± 10.4 to 120.7 ± 10.7 mmHg, mean ± SD, P

Acrylamide (AA) is a heat-induced food contaminant considered as genotoxic carcinogen. The present study investigated the influence of nutritional and lifestyle preferences on human AA exposure. A 10-day human study was performed with ten volunteers without nutritional preferences (omnivores) and ten vegans. Volunteers self-reported their daily routine and dietary habits. Overall mean AA intake, calculated from contents of diet duplicates, was 0.32 ± 0.19 µg/kg body weight (bw)/day with marked inter-day and inter-volunteer variabilities. Vegans ingested more AA (0.38 ± 0.23 µg/kg bw/day) than omnivore volunteers without dietary restrictions (0.26 ± 0.10 µg/kg bw/day). Excretion kinetics of urinary AA-related mercapturic acids N-acetyl-S-(2-carbamoylethyl)-L-cysteine and N-acetyl-S-(2-hydroxy-2-carbamoylethyl)-L-cysteine were essentially concordant with the respective dietary AA intake. Disproportionately enhanced AA-related biomarker excretion could be traced back to reportedly inadvertent, passive exposure to tobacco and/or fire smoke, as evidenced by the respective urinary exposure biomarkers, cotinine and N-acetyl-S-(2-cyanoethyl)-L-cysteine. Although the study is based on the comparison of small volunteer groups, the results confirm the association of AA exposure biomarkers with documented dietary preferences and lifestyle factors. Some additional contribution of endogenous background AA exposure was demonstrated individually. Disproportionately enhanced AA exposure is suggested to result from passive exposure to tobacco and/or barbecue smoke.

Processed Animal Proteins (PAPs) are considered as a sustainable protein source to improve the nutritional profile of feed for livestock and aquaculture. However, the use of these proteins is strongly regulated since the bovine spongiform encephalopathy (BSE) crisis. The reintroduction of nonruminant PAPs for use in aquaculture in 2013 has driven the need for alternative analytical methods to determine the species origin as well as the tissue source (legal or not). The current official methods, light microscopy and polymerase chain reaction, do not fulfill these requirements. Furthermore, future methods need to be quantitative, because the pending zero-tolerance-concept is planned to be replaced by accurate thresholds. Here, we developed a 7-plex mass spectrometry-based immunoassay that is capable of quantifying 0.1% (w/w) ruminant PAP in feed in a tissue- and species-specific way. The workflow comprises a 2 h tryptic digestion of PAPs in suspension, an immunoaffinity enrichment of peptides, and LC-MS/MS-based quantification. In combination with a previously published assay for species identification, we were able to confirm the species and tissue origin of six ring trial samples obtained in former PCR and microscopy proficiency tests. The sensitive, quantitative, species- and tissue-specific character of the developed assays meets the requirements for new methods for PAP detection and can be used in future feed authentication studies.

Carcinogenic benzo[a]pyrene (BP) and other non-carcinogenic polycyclic aromatic hydrocarbons (PAH) like fluoranthene (FA) and pyrene (PYR) occur as food contaminants. Molecular effects of BP, FA and PYR in human liver cells were investigated using mixtures occurring in grilled meat. Activation of aryl hydrocarbon receptor (AHR) and constitutive androstane receptor (CAR) was investigated along with target gene expression. Mixture effects on BP metabolite profile and DNA-damaging potential were studied as biological downstream effects. Compared to BP, FA and PYR activated the AHR only weakly. Mixtures were less efficient than BP. Analysis of CYP1A1 expression showed synergistic induction after co-exposure in HepaRG cells. FA and PYR were strong CAR agonists, whereas BP was less potent. Mixtures containing BP caused a strong decrease of CAR transactivation in line with lower CYP2B6 expression. The BP metabolite profile and BP-induced DNA damage were only weakly affected. PAH mixtures modulate AHR, CAR activation and their target genes. However, these mixture effects appear not to be reflected at the level of downstream events like BP metabolite formation or BP-induced DNA damage. Our study clearly shows that endpoints at all biological levels should be considered for mixture evaluation, instead of drawing conclusions exclusively based on early molecular events.

Tebuconazole, a member of the triazole group of fungicides, exerts hepatotoxicity in rodent studies. Knowledge on the molecular mechanisms underlying tebuconazole toxicity is limited. Previous studies suggest that activation of xenobiotic-sensing nuclear receptors plays a role in triazole fungicide-mediated hepatotoxicity. This study aimed to characterize the ability of tebuconazole to activate gene expression via the aryl hydrocarbon receptor (AHR). Results demonstrate a statistically significant induction of the AHR target genes CYP1A1 and CYP1A2 in HepG2 and HepaRG human liver cells in vitro at concentrations corresponding to tebuconazole tissue levels reached under subtoxic conditions in vivo. CYP1A1 and CYP1A2 induction was abolished in the presence of an AHR antagonist or in AHR-knockout HepaRG cells, substantiating the importance of the AHR for the observed effects. Although the results indicate that tebuconazole is a weak inducer of AHR-dependent genes, combined exposure of HepaRG cells to tebuconazole and the previously identified AHR agonist propiconazole showed additive effects on CYP1A1 and CYP1A2 expression. In summary, we demonstrate that AHR-downstream gene expression is affected by tebuconazole in an AHR-dependent manner. Data indicate that dose addition may be assumed for the assessment of AHR-related effects of triazole fungicide mixtures.

Pyrrolizidine alkaloids (PA) are a group of secondary plant metabolites belonging to the most widely distributed natural toxins. PA intoxication of humans leads to severe liver damage, such as hepatomegaly, hepatic necrosis, fibrosis and cirrhosis. An acute consequence observed after ingestion of high amounts of PA is veno-occlusive disease (VOD) where the hepatic sinusoidal endothelial cells are affected. However, the mechanisms leading to VOD after PA intoxication remain predominantly unknown. Thus, we investigated PA-induced molecular effects on human umbilical vein endothelial cells (HUVEC). We compared the effects of PA with the effects of PA metabolites obtained by in vitro metabolism using liver homogenate (S9 fraction). In vitro-metabolized lasiocarpine and senecionine resulted in significant cytotoxic effects in HUVEC starting at 300 μM. Initial molecular effect screening using a PCR array with genes associated with endothelial cell biology showed PA-induced upregulation of the Fas receptor, which is involved in extrinsic apoptosis, and regulation of a number of interleukins, as well as of different enzymes relevant for prostanoid synthesis. Modulation of prostanoid synthesis was subsequently studied at the mRNA and protein levels and verified by increased release of prostaglandin I2 as the main prostanoid of endothelial cells. All effects occurred only with in vitro-metabolically activated PA lasiocarpine and senecionine. By contrast, no effect was observed for the PA echimidine, heliotrine, lasiocarpine, senecionine, senkirkine and platyphylline in the absence of an external metabolizing system up to the highest tested concentration of 500 μM. Overall, our results confirm the metabolism-dependent toxification of PA and elucidate the involved pathways. These include induction of inflammatory cytokines and deregulation of the prostanoid synthesis pathway in endothelial cells, linking for the first time PA-dependent changes in prostanoid release to distinct alterations at the mRNA and protein levels of enzymes of prostanoid synthesis.

International audience; Aluminum (Al) can be taken up from food, packaging, or the environment and thus reaches the human gastrointestinal tract. Its toxic potential after oral uptake is still discussed. The fate of different solid and ionic Al species during the passage through the digestive tract is the focus of this research, as well as the cellular effects caused by these different Al species. The present study combines the physicochemical processing of three recently studied Al species (metallic Al0, mineral Al2O3, and soluble AlCl3) in artificial digestion fluids with in vitro cell systems for the human intestinal barrier. Inductively coupled plasma mass spectrometry (ICP-MS) and small-angle X-ray scattering (SAXS) methods were used to characterize the Al species in the artificial digestion fluids and in cell culture medium for proliferating and differentiated intestinal Caco-2 cells. Cytotoxicity testing and cellular impedance measurements were applied to address the effects of digested Al species on cell viability and cell proliferation. Microarray-based transcriptome analyses and quantitative real-time PCR were conducted to obtain a deeper insight into cellular mechanisms of action and generated indications for cellular oxidative stress and an influence on xenobiotic metabolism, connected with alterations in associated signaling pathways. These cellular responses, which were predominantly caused by formerly ionic Al species and only at very high concentrations, were not impacted by artificial digestion. A two-directional conversion of Al between ionic species and solid particles occurred throughout all segments of the gastrointestinal tract, as evidenced by the presence of nanoscaled particles. Nevertheless, this presence did not increase the toxicity of the respective Al species.

Pyrrolizidine alkaloids (PA) are secondary plant metabolites that occur as food and feed contaminants. Acute and subacute PA poisoning can lead to severe liver damage in humans and animals, comprising liver pain, hepatomegaly and the development of ascites due to occlusion of the hepatic sinusoids (veno-occlusive disease). Chronic exposure to low levels of PA can induce liver cirrhosis and liver cancer. However, it is not well understood which transcriptional changes are induced by PA and whether all hepatotoxic PA, regardless of their structure, induce similar responses. Therefore, a 28-day subacute rat feeding study was performed with six structurally different PA heliotrine, echimidine, lasiocarpine, senecionine, senkirkine, and platyphylline, administered at not acutely toxic doses from 0.1 to 3.3 mg/kg body weight. This dose range is relevant for humans, since consumption of contaminated tea may result in doses of ~ 8 µg/kg in adults and cases of PA ingestion by contaminated food was reported for infants with doses up to 3 mg/kg body weight. ALT and AST were not increased in all treatment groups. Whole-genome microarray analyses revealed pronounced effects on gene expression in the high-dose treatment groups resulting in a set of 36 commonly regulated genes. However, platyphylline, the only 1,2-saturated and, therefore, presumably non-hepatotoxic PA, did not induce significant expression changes. Biological functions identified to be affected by high-dose treatments (3.3 mg/kg body weight) comprise cell-cycle regulation associated with DNA damage response. These functions were found to be affected by all analyzed 1,2-unsaturated PA. In conclusion, 1,2-unsaturated hepatotoxic PA induced cell cycle regulation processes associated with DNA damage response. Similar effects were observed for all hepatotoxic PA. Effects were observed in a dose range inducing no histopathological alterations and no increase in liver enzymes. Therefore, transcriptomics studies identified changes in expression of genes known to be involved in response to genotoxic compounds at PA doses relevant to humans under worst case exposure scenarios. Electronic supplementary material The online version of this article (10.1007/s00204-020-02779-2) contains supplementary material, which is available to authorized users.

2-monochloropropanediol (2-MCPD), 3-monochloropropanediol (3-MCPD) and their fatty acid esters have recently been identified as heat-induced contaminants in fat- and salt-containing foodstuff. Toxicity of 3-MCPD has been studied previously in some detail. Disturbance of glycolysis and cellular redox functions appear to be involved in 3-MCPD toxicity. By contrast, only very few toxicological data are available for 2-MCPD or 2-MCPD esters, especially at the molecular level. This study was therefore aimed to provide a comprehensive overview of proteomic alterations induced in rat kidney and liver by 2-MCPD and 2-MCPD dipalmitate, a representative 2-MCPD fatty acid ester. Sub-toxic doses of 10 mg/kg body weight 2-MCPD, or equimolar doses of 2-MCPD dipalmitate were applied in a 28-day in vivo gavage oral toxicity study in male rats. Two-dimensional gel electrophoresis and mass-spectrometric protein identification using material from 5 animals per treatment group were employed together with bioinformatic data mining to obtain information about the molecular basis of the observed proteomic alterations. Obtained data indicate toxic consequences of 2-MCPD exposure in the kidney and provide evidence that 2-MCPD exerts its cellular effects in rat kidney by mechanisms different from 3-MCPD.

International audience; Aluminum (Al) is one of the most common elements in the earth crust and increasingly used in food, consumer products and packaging. Its hazard potential for humans is still not completely understood. Besides the metallic form, Al also exists as mineral, including the insoluble oxide, and in soluble ionic forms. Representatives of these three species, namely a metallic and an oxidic species of Al-containing nanoparticles and soluble aluminum chloride, were applied to human intestinal cell lines as models for the intestinal barrier. We characterized physicochemical particle parameters, protein corona composition, ion release and cellular uptake. Different in vitro assays were performed to determine potential effects and molecular modes of action related to the individual chemical species. For a deeper insight into signaling processes, microarray transcriptome analyses followed by bioinformatic data analysis were employed. The particulate Al species showed different solubility in biological media. Metallic Al nanoparticles released more ions than AlO nanoparticles, while AlCl showed a mixture of dissolved and agglomerated particulate entities in biological media. The protein corona composition differed between both nanoparticle species. Cellular uptake, investigated in transwell experiments, occurred predominantly in particulate form, whereas ionic Al was not taken up by intestinal cell lines. Transcellular transport was not observed. None of the Al species showed cytotoxic effects up to 200 µg Al/mL. The transcriptome analysis indicated mainly effects on oxidative stress pathways, xenobiotic metabolism and metal homeostasis. We have shown for the first time that intestinal cellular uptake of Al occurs preferably in the particle form, while toxicological effects appear to be ion-related.

With the reintroduction of nonruminant processed animal proteins (PAPs) for use in aquaculture in 2013, there is a suitable alternative to replace expensive fish meal in fish feed. Nevertheless, since the bovine spongiform encephalopathy (BSE) crisis, the use of PAPs in feed is strictly regulated. To date, light microscopy and polymerase chain reaction are the official methods for proving the absence of illegal PAPs in feed. Due to their limitations, alternative methods for the quantitative species differentiation are needed. To address this issue, we developed and validated an 8-plex mass spectrometry-based immunoassay. The workflow comprises a tryptic digestion of PAPs and blood products in suspension, a cross-species immunoaffinity enrichment of 8 species-specific alpha-2-macroglobulin peptides using a group-specific antibody, and a subsequent analysis by ultrahigh-performance liquid chromatography coupled to tandem mass spectrometry for species identification and quantification. This workflow can be used to quantitatively determine the species origin in future feed authentication studies.

A real-time PCR method was developed for the detection of black soldier fly (Hermetia illucens) in feed. The method amplifies a sequence of 89 bp size within the mitochondrial barcode region (cytochrome c oxidase gene, COI). The PCR efficiency of the system achieved ∼96% in a background of commercial fish feed DNA. The Limit of Detection (LOD6) of the method with pure Hermetia illucens DNA diluted in compound feed DNA is 0.1 genome copies, corresponding to an absolute amount of 0.13 pg DNA. In addition, the sensitivity was investigated with differently processed material. It could be shown that the method reliably detects delipidated Hermetia protein in mixtures down to 0.01% (w/w) in aquaculture feed. Supplementary experiments exploring the impact of drying, heating and defatting revealed no negative impact on the sensitivity even for hot solvent-extracted dried larvae pre-treated at 140 °C/20 min. The specificity of the PCR system was confirmed with a broad range of terrestrial and aqueous species. The selection included most relevant arthropods, comprising the seven insect species, which are allowed in aquaculture feeding in Europe. It is concluded that the method is robust and fit for purpose, enabling the sensitive and reliable detection of raw or processed Hermetia illucens products as an upcoming new component in feed but also food. Since a standard time/temperature program is used the approach is well suited for the development of future multiplex or relative quantitation methods.

TiO 2 nanomaterials (NM) have a wide range of industrial applications, including their use in food products. The incorporation of these NMs in consumer products represents a clear concern for public health safety agencies and consumers, and further investigation of the potential impact of these products on human health is necessary. Indeed, since human oral exposure to TiO 2 NMs is expected to increase in the years to come, there exist legitimate concerns about the risk assessment of these nanomaterials present in food products. A considerable amount of studies investigating the adverse effects of TiO 2 NMs have focused on the genotoxic effects of these NMs, and more recently they have been classified by the International Agency for Research on Cancer (IARC) as carcinogen group 2B following inhalation studies (IARC, 2010). While numerous data are available for anatase or mixes of anatase/rutile forms, the toxicity and the genotoxicity of rutile TiO 2 NMs have been rarely investigated. The aim of our study was therefore to investigate the cytotoxic and genotoxic effects of two rutile TiO 2 NMs, differing in surface coating, NM103 (hydrophobic) and NM104 (hydrophilic), on intestinal and hepatic cell models. Following 3 or 24 h treatments with concentrations of TiO 2 NMs from 1.2 to 80 μg/cm 2 , we have assessed the genotoxicity of these NMs with γH2AX, alkaline comet assay and micronucleus (MN) assays. Cellular viability and effects on oxidative stress were also evaluated. Although TEM imaging demonstrated the presence of the two TiO 2 NMs within the cytoplasm, no significant cytotoxic or genotoxic were observed in either cell model. We have also evaluated and taken into account a variety of potential sources of interference of NMs with cellular assays. TiO 2 NMs present in the cytoplasm introduce uncertainty in the scoring of micronuclei, and therefore this assay is not recommended for the evaluation of the genotoxicity of TiO 2 NMs, or other NMs demonstrating similar interference. The unique properties of TiO 2 NMs introduce additional complexity for genotoxicity testing, and caution must be taken in order to obtain reliable results necessary for accurate hazard assessment.

2- and 3-monochloropropanediol (2-MCPD) and their fatty acid esters are food contaminants which are concomitantly formed upon thermal treatment of foodstuff containing fats and salt. Exposure to 2- or 3-MCPD thus results, for example, from refined vegetable oils, in instant meals or infant formula, as well as in cereals or pastries. The molecular mechanisms of 2-MCPD toxicity are poorly understood. Here, we performed a comprehensive proteomic analysis of 2-MCDP-induced alterations in the testes from rats following oral administration of 10 mg/kg body weight per day 2-MCPD, or an equimolar dose of 2-MCPD dipalmitate as a representative 2-MCPD fatty acid ester. In the absence of overt histopathologically detectable toxicity, moderate alterations in cellular proteomic signatures were recorded. The observations are in line with the assumption that the molecular mechanisms of 2-MCPD and 3-MCPD toxicity differ. Observed proteomic alterations point towards effects of 2-MCPD on mitogen-dependent signaling and mitochondrial energy utilization. Presented data for the first time provide insight into proteomic effects of 2-MCPD in testicular tissue.

Silver nanoparticles are among the most widely used and produced nanoparticles. Because of their frequent application in consumer products, the assessment of their toxicological potential has seen a renewed importance. A major difficulty is the traceability of nanoparticles in in vitro and in vivo experiments. Even if the particles are labeled, for example, by a fluorescent marker, the dynamic exchange of ligands often prohibits their spatial localization. Our study provides an insight into the adsorption and desorption kinetics of two different fluorescent labels on silver nanoparticles with a core radius of 3 nm by dynamic light scattering, small-angle X-ray scattering, and fluorescence spectroscopy. We used BSA-FITC and tyrosine as examples for common fluorescent ligands. It is shown that the adsorption of BSA-FITC takes at least 3 days, whereas tyrosine adsorbs immediately. The quantitative amount of stabilizer on the particle surface was determined by fluorescence spectroscopy and revealed that the particles are stabilized by a monolayer of BSA-FITC (corresponding to 20 ± 9 molecules), whereas tyrosine forms a multilayered structure consisting of 15900 ± 200 molecules. Desorption experiments show that the BSA-FITC-stabilized particles are ideally suited for application in in vitro and in vivo experiments because the ligand desorption takes several days. Depending on the BSA concentration in the particles surroundings, the rate constant is k = 0.2 per day or lower when applying first order kinetics, that is, 50% of the BSA-FITC molecules are released from the particle's surface within 3.4 days. For illustration, we provide a first application of the fluorescence-labeled particles in an uptake study with two different commonly used cell lines, the human liver cell model HepG2 and the human intestinal cell model of differentiated Caco-2 cells.

DINCH® (di-isononyl cyclohexane-1,2-dicarboxylate) is a non-phthalate plasticizer that has been developed to replace phthalate plasticizers such as DEHP (di-2-ethylhexyl phthalate) or DINP (di-isononyl phthalate). DINCH® is metabolized to its corresponding monoester and subsequently to oxidized monoester derivatives. These are conjugated to glucuronic acid and subject to urinary excretion. In contrast to DINCH®, there are almost no toxicological data available regarding its primary and secondary metabolites. The present study aimed at the characterization of potential endocrine properties of DINCH® and five DINCH® metabolites by using reporter gene assays to monitor the activity of the human nuclear receptors ERα, ERβ, AR, PPARα and PPARγ in vitro. DINCH® itself did not have any effect on the activity of these receptors whereas DINCH® metabolites were shown to activate all these receptors. In the case of AR, DINCH® metabolites predominantly enhanced dihydrotestosterone-stimulated AR activity. In the H295R steroidogenesis assay, neither DINCH® nor any of its metabolites affected estradiol or testosterone synthesis. In conclusion, primary and secondary DINCH® metabolites exert different effects at the molecular level compared to DINCH® itself. All these in vitro effects of DINCH® metabolites, however, were only observed at high concentrations such as 10 μM or above which is about three orders of magnitude above reported DINCH® metabolite concentrations in human urine. Thus, the in vitro data do not support the notion that DINCH® or any of the investigated metabolites may exert considerable endocrine effects in vivo at relevant human exposure levels.

Pyrrolizidine alkaloids (PAs) comprise a large group of more than 660 secondary metabolites found in more than 6000 plant species worldwide. Acute PA intoxication induces severe liver damage. Chronic exposure to sub-lethal doses may cause cumulative damage or cancer. Nuclear receptor activation often constitutes a molecular event for xenobiotic-induced toxicity. However, so far nothing is known about potential interactions of PAs with nuclear receptors as a toxicological mode of action. Thus, in the present study PA-dependent activation of a comprehensive panel of nuclear receptors (PPARs, LXRα, RARα, RXRα, FXR, CAR, PXR, ERα/β) was investigated using GAL4/UAS-based transactivation reporter gene assays. To cover the most frequently occurring PA structure types (retronecine, heliotridine and otonecine type; as well as monoester, open-chain diester and cyclic diester) different PAs were analyzed for interaction with nuclear receptors. Most of the nuclear receptors investigated were not affected by the tested PAs. However, significant activation was found for PXR, which was exclusively activated by the open-chain diesters, echimidine and lasiocarpine. Induction of the model PXR target gene CYP3A4 by PAs was verified at the mRNA, protein and enzyme activity level. In conclusion, PXR activation and PXR-mediated induction of CYP3A4 expression by PAs seem to be structure-dependent. Data suggest that only open-chain diesters act as PXR agonists. This might imply that a PXR-mediated mode of action may contribute to the hepatotoxicity of PAs that is dependent on PA structure.

Pyrrolizidine alkaloids (PA) are secondary plant compounds. PA intoxication in humans causes severe acute and chronic hepatotoxicity. However, the molecular mechanisms of PA hepatotoxicity in humans are not well understood yet. Therefore, we investigated cell death parameters in human HepaRG cells following either single (24 h) or repeated dose treatment (14 d) with structurally different PA of the retronecine (echimidine, senecionine), heliotridine (heliotrine), and otonecine type (senkirkine). After 24 h of exposure only retronecine-type PA were cytotoxic in HepaRG cells and induced apoptosis indicated by a loss of membrane asymmetry, disruption of the mitochondrial membrane potential, and increased pro-caspase and PARP cleavage. In contrast, after 14 d all four PA exerted the aforementioned effects. Furthermore, the apoptotic events caspase 3, 8 and 9 activation as well as nuclear condensation and DNA fragmentation were only detected for the retronecine-type PA after single exposure (6 h). Overall, our studies revealed a time- and structure-dependent apoptosis after PA exposure, suggesting that retronecine-type PA seem to be more potent apoptosis inducers than heliotridine- or otonecine-type PA. Furthermore, our results suggest that PA-induced apoptosis in HepaRG cells occur most probably by involving both, the extrinsic death receptor pathway as well as the intrinsic mitochondrial pathway.

An artificial digestion of silver nitrate is reported. It is shown that AgSCN nanoparticles emerge from ionic silver in saliva and remain present during the entire digestion process. The particles were characterized by infrared spectroscopy and small- and wide-angle X-ray scattering (SAXS/WAXS) regarding their composition and size distribution.

Consumers are orally exposed to nanoparticulate or soluble species of the non-essential element silver due to its use in food contact materials or as a food additive. Potential toxicity of silver nanoparticles has gained special scientific attention. A fraction of ingested ionic or particulate silver is taken up in the intestine and transported to the liver, where it may induce oxidative stress and elicit subsequent adverse responses. Here, we present a comprehensive analysis of global proteomic changes induced in human Hep G2 hepatocarcinoma cells by different concentrations of AgPURE silver nanoparticles or by corresponding concentrations of ionic silver. Bioinformatic analysis of proteomic data confirms and substantiates previous findings on silver-induced alterations related to redox stress, mitochondrial dysfunction, intermediary metabolism, inflammatory responses, posttranslational protein modification and other cellular parameters. Similarities between the effects exerted by the two silver species are in line with the assumption that silver ions released from nanoparticles substantially contribute to their toxicity. Moreover, a comparative bioinformatic evaluation of proteomic effects in hepatic and intestinal cells exerted either by silver nanoparticles or bionic silver is presented. Our results show that, despite remarkable differences at the level of affected proteins in the different cell lines, highly similar biological consequences, corresponding to previous in vivo findings, can be deduced by applying appropriate bioinformatic data mining.

The elucidation of mechanisms underlying the cellular uptake of nanoparticles (NPs) is an important topic in nanotoxicological research. Most studies dealing with silver NP uptake provide only qualitative data about internalization efficiency and do not consider NP-specific dosimetry. Therefore, we performed a comprehensive comparison of the cellular uptake of differently coated silver NPs of comparable size in different human intestinal Caco-2 cell-derived models to cover also the influence of the intestinal mucus barrier and uptake-specialized M-cells. We used a combination of the Transwell system, transmission electron microscopy, atomic absorption spectroscopy, and ion beam microscopy techniques. The computational in vitro sedimentation, diffusion, and dosimetry (ISDD) model was used to determine the effective dose of the particles in vitro based on their individual physicochemical characteristics. Data indicate that silver NPs with a similar size and shape show coating-dependent differences in their uptake into Caco-2 cells. The internalization of silver NPs was enhanced in uptake-specialized M-cells while the mucus did not provide a substantial barrier for NP internalization. ISDD modeling revealed a fivefold underestimation of dose-response relationships of NPs in in vitro assays. In summary, the present study provides dosimetry-adjusted quantitative data about the influence of NP coating materials in cellular uptake into human intestinal cells. Underestimation of particle effects in vitro might be prevented by using dosimetry models and by considering cell models with greater proximity to the in vivo situation, such as the M-cell model.

The breadth of applications of nanoparticles and the access to food-associated consumer products containing nanosized materials lead to oral human exposure to such particles. In biological fluids nanoparticles dynamically interact with biomolecules and form a protein corona. Knowledge about the protein corona is of great interest for understanding the molecular effects of particles as well as their fate inside the human body. We used a mass spectrometry-based toxicoproteomics approach to elucidate mechanisms of toxicity of silver nanoparticles and to comprehensively characterize the protein corona formed around silver nanoparticles in Caco-2 human intestinal epithelial cells. Results were compared with respect to the cellular function of proteins either affected by exposure to nanoparticles or present in the protein corona. A transcriptomic data set was included in the analyses in order to obtain a combined multiomics view of nanoparticle-affected cellular processes. A relationship between corona proteins and the proteomic or transcriptomic responses was revealed, showing that differentially regulated proteins or transcripts were engaged in the same cellular signaling pathways. Protein corona analyses of nanoparticles in cells might therefore help in obtaining information about the molecular consequences of nanoparticle treatment.

The lipophilic marine biotoxin okadaic acid (OA) represents a natural contaminant produced by algae accumulating in seafood. Acute intoxications result in diarrhetic shellfish poisoning causing symptoms like nausea, vomiting and abdominal cramps. OA was preincubated with liver enzymes present in S9 fractions from humans, rats and rats pretreated with enzyme inducers in the presence or absence of the cofactor NADPH to investigate hepatic metabolism. Cytotoxicity was examined in HepG2 cells and metabolites of OA were determined by LC-MS/MS. Strong cytotoxicity was observed in HepG2 cells treated with OA that was preincubated in S9 fractions without NADPH. However, neither metabolites nor a decrease of OA itself were found. The addition of NADPH to the S9 fractions of rats resulted in a decreased cytotoxicity of OA, but a stronger toxicity in HepG2 cells was observed from OA preincubated in human S9 fractions with NADPH. Metabolite profiles of each S9 mix revealed that higher amounts of detoxified metabolites were formed by NADPH-dependent enzymes of rats compared to the same enzymes of humans. These differences in OA detoxification by NADPH-dependent liver enzymes of rats and humans may be of significance in the extrapolation of toxicological data from animal models (rats to humans, for example).

Fatty acid esters of glycidol (glycidyl esters) are processing contaminants generated as a byproduct of the industrial deodorization of vegetable oils and fats. Oral intake of glycidyl esters leads to the release of glycidol in the gastrointestinal tract. Glycidol is carcinogenic, genotoxic and teratogenic in rodents. It is rated as probably carcinogenic to humans (IARC group 2A). The determination of internal exposure of glycidol may support the assessment of the possible human health risks related to glycidyl ester intake. For this purpose, hemoglobin adducts of glycidol may be suitable biomarkers reflecting the cumulative exposure of up to four months. We applied a modified Edman degradation to assess the glycidol adduct at the N-terminal valine, N-(2,3-dihydroxypropyl)-valine (2,3-diHOPr-Val), of hemoglobin. The modified valine was cleaved with fluorescein-5-isothiocyanate (FITC), resulting in the formation of N-(2,3-dihydroxypropyl)-valine fluorescein thiohydantoin (DHP-Val-FTH). An isotope-dilution technique was developed for the quantification of the thiohydantoin analyte by ultra performance liquid chromatography-tandem mass spectrometry (UPLC–MS/MS) and DHP-Val-d7-FTH as reference standard. The limit of detection was 4 fmol DHP-Val-FTH per injection corresponding to 0.7 pmol 2,3-diHOPr-Val/g hemoglobin. The adduct levels in blood samples of 12 non-smoking participants were in the range of 2.2–4.9 pmol 2,3-diHOPr-Val/g hemoglobin. The current work presents the first isotope-dilution technique using UPLC–MS/MS for the quantification of 2,3-diHOPr-Val at the N-terminus of hemoglobin as a sensitive and convenient alternative to earlier GC–MS methods.

Furfuryl alcohol is a common food contaminant, which is formed by acid- and heat-catalyzed degradation of fructose and glucose. Its carcinogenic effect in rodents originates most likely from sulfotransferase (SULT)-catalyzed conversion into the mutagenic sulfate ester 2-sulfoxymethylfuran. In this study, a protein adduct biomarker was sought for the medium-term internal exposure to furfuryl alcohol. A UPLC-MS/MS screening showed that the adduct N-((furan-2-yl)methyl)-Val (FFA-Val) at the N-terminus of hemoglobin is a valid target analyte. The Val cleavage by fluorescein isothiocyanate-mediated Edman degradation yielded 3-fluorescein-1-(furan-2-ylmethyl)-5-(propan-2-yl)-2-thioxoimidazolidin-4-one (FFA-Val-FTH), which was characterized by 1H and 13C NMR spectroscopy. An isotope-dilution method for the quantification of FFA-Val-FTH by UPLC-MS/MS was developed. It was used to study the adduct formation in furfuryl alcohol-treated FVB/N mice and the influence of ethanol and the alcohol dehydrogenase (ADH) inhibitor 4-methylpyrazole on the adduct levels. The administration of 400 mg/kg body weight furfuryl alcohol alone led to 12.5 and 36.7 pmol FFA-Val/g Hb in blood samples of male and female animals, respectively. The co-administration of 1.6 g ethanol/kg body weight increased FFA-Val levels by 1.4-fold in males and by 1.5-fold in females. The co-administration of 100 mg 4-methylpyrazole/kg body weight had a similar effect on the adduct levels. A high correlation was observed between adduct levels in hemoglobin and in hepatic DNA samples determined in the same animal experiment. This indicated that FFA-Val is a valid biomarker for the internal exposure to 2-sulfoxymethylfuran, which may be suitable to monitor furfuryl alcohol exposure also in humans.

Ingestion of fish may exert severe health effects in food-allergic individuals. Sensitive analytical methods are required for the surveillance of the obligation to label fish as a potentially allergenic ingredient in foodstuff. Recently a sensitive multi-copy PCR method has been published to detect a broad spectrum of fish species by amplification of the mitochondrial 12S rRNA gene. The performance of this method in real-life samples also containing DNA from other sources such as plants or animals has not been studied yet. The applicability domain of the PCR, especially for allergen detection, is therefore still unclear. Here we assessed the ability of the PCR system to detect fish in a background of excess animal and plant DNA. The results demonstrate satisfying performance with various fish species but pronounced PCR inhibition by swine DNA. Subsequent analyses revealed homology of one PCR primer to the swine 12S rRNA gene, causing competition during the PCR reaction and thus raising the limit of detection by two orders of magnitude. In conclusion, the tested PCR system is suited for fish detection in plant matrices, but not in matrices containing excess animal, especially swine, DNA. Application of the PCR system for allergen detection is therefore not recommended.

Wahrend der Herstellung und Zubereitung von Lebensmitteln konnen bei hohen Temperaturen unerwunschte Verbindungen mit gesundheitsschadlichem Potenzial entstehen. Zu den am besten untersuchten erhitzungsbedingten Kontaminanten zahlen Acrylamid, Furan sowie die Fettsaureester von Glycidol und Monochlorpropandiolen. Dieser Beitrag stellt die wesentlichen Erkenntnisse zur Entstehung, zur Toxikologie und zur Exposition dieser Verbindungen dar. Acrylamid und Glycidol haben sich in tierexperimentellen Untersuchungen als kanzerogen mit gentoxischem Mechanismus erwiesen; ihre Gehalte in Lebensmitteln sollten minimiert werden. Fur 3‑Monochlorpropandiol (3-MCPD) kann eine tolerierbare tagliche Aufnahmedosis abgeleitet werden. Dagegen ist fur Furan und 2‑MCPD aufgrund der ungenugenden Datenlage eine abschliesende Risikobewertung derzeit noch nicht moglich. Zusatzlich zu den genannten Verbindungen wurde eine Vielzahl weiterer erhitzungsbedingter Substanzen in Lebensmitteln identifiziert, zu denen zumeist keine Daten zur Toxikologie und Exposition vorliegen. Daher kann fur diese Verbindungen gegenwartig keine Risikobewertung vorgenommen werden. Fur eine Priorisierung dieser grosen Anzahl an Verbindungen nach ihrem moglichen Gefahrdungspotenzial erscheint zunachst eine Nutzung von Computermodellen sinnvoll, die anhand der chemischen Struktur einer Verbindung eine Vorhersage zu definierten toxikologischen Endpunkten erlaubt. Fur Substanzen, die als prioritar eingestuft worden sind, sind jedoch fur eine aussagekraftige Risikobewertung weitere, zumeist kosten- und zeitintensive Untersuchungen bezuglich der Toxikologie und der Quantifizierung dieser Verbindungen in Lebensmitteln notwendig.

Nanomaterialien weisen in mindestens einer Dimension Ausmase von unter 100 nm auf. Grundsatzlich werden organische und anorganische Nanopartikel unterschieden. Nanoskalige Strukturen konnen sich auserdem in ihrer Form, der chemischen Natur und den physikalischen Parametern stark unterscheiden und sowohl auf naturlichem Wege entstehen als auch bewusst technisch hergestellt werden. Gegenstand aktueller toxikologischer Forschung sind mogliche spezielle toxikologische Eigenschaften von Nanopartikeln, die auf einer, im Vergleich zu groseren Partikeln erhohten Reaktivitat infolge eines vergroserten Verhaltnisses von Oberflache zu Volumen oder aus einer bevorzugten Aufnahme in Zellen und Gewebe resultieren konnten. Verbraucher konnen durch Lebensmittel uber die orale Aufnahmeroute gegenuber Nanomaterialien exponiert sein, die naturlich entstanden, unbeabsichtigt hineingelangt oder bewusst zugesetzt wurden. Im Rahmen dieser Arbeit soll ein Uberblick uber das Vorkommen von Nanomaterialien in Lebensmitteln sowie uber deren gesetzliche Definition und Kennzeichnungspflicht gegeben werden. Des Weiteren werden spezifische Charakteristika der oralen Aufnahme von Nanomaterialien besprochen und Herausforderungen bei der experimentellen Untersuchung der gastrointestinalen Aufnahme und Wirkung von nanoskaligen Partikeln zusammen mit moglichen toxikologisch relevanten Eigenschaften und Wirkmechanismen beleuchtet. Ungeachtet einer Vielzahl von verfugbaren experimentellen Studien bestehen noch Wissenslucken hinsichtlich der Exposition gegenuber oral aufgenommener Nanopartikel sowie hinsichtlich moglicher toxischer Wirkungen solcher Partikel. Diese mussen geschlossen werden, um die Risikobewertung solcher Materialien in Lebensmitteln und ihren Verpackungen zu ermoglichen.

The assessment of health risks resulting from the intake of genotoxic carcinogens in food depends essentially on a valid exposure assessment. The reliability of the external exposure estimation is restricted by various factors, e. g. inaccurate data from dietary protocols and variations of food contaminant contents. As an alternative, the individual internal exposure to genotoxic substances may be described by specific biomarkers in different matrices. For example, mercapturic acids formed after glutathione conjugation of electrophilic metabolites can be detected in the urine. This typically reflects the exposure to the parent compound over a period of one to two days. The determination of adducts in the blood proteins serum albumin (SA) and hemoglobin (Hb) allows for conclusions to be drawn about the external exposure within the last three weeks (SA) or within the last four months (Hb). Protein adducts are used routinely in occupational medicine as biomarkers of internal exposure to substances in the ambient air of the workplace. The availability of increasingly sensitive analytical techniques also makes it possible to detect numerous adducts in proteins from human blood samples that are formed after the continuous intake of very small doses of toxic substances from foods. Here, we present the current state of science exemplified by protein adducts of the food contaminants acrylamide, aflatoxin B1 and glycidol. The biomarker can be used in the future to investigate previously unknown relationships between internal exposure and disease incidences.

Iron oxides used as food colorants are listed in the European Union with the number E172. However, there are no specifications concerning the fraction of nanoparticles in these pigments. Here, seven E172 products were thoroughly characterized. Samples of all colors were analyzed with a broad spectrum of methods to assess their physico-chemical properties. Small-Angle X-ray Scattering (SAXS), Dynamic Light Scattering (DLS), Transmission Electron Microscopy (TEM), zeta-potential, Inductively Coupled Plasma-Mass Spectrometry (ICP-MS), X-ray diffraction (XRD), Brunauer-Emmett-Teller analysis (BET), Asymmetric Flow Field-Flow Fractionation (AF4) and in vitro cell viability measurements were used. Nanoparticles were detected in all E172 samples by TEM or SAXS measurements. Quantitative results from both methods were comparable. Five pigments were evaluated by TEM, of which four had a size median below 100 nm, while SAXS showed a size median below 100 nm for six evaluated pigments. Therefore, consumers may be exposed to iron oxide nanoparticles through the consumption of food pigments.

The author would like to thank N. Bakhiya, S. Hessel-Pras, B. Sachse, and B. Dusemund for their support in the chapter about pyrrolizidine alkaloids.

Humans are exposed to pesticide residues through various food products. As these residues can occur in mixtures, there is a need to investigate possible mixture effects on human health. Recent exposure studies revealed the preponderance of imazalil, thiacloprid, and clothianidin in food diets. In this study, we assessed their toxicity alone and in binary mixtures in a 28-day gavage study in female Wistar rats. Five dose levels (up to 350 mg/kg bw/day) ranging from a typical toxicological reference value to a clear effect dose were applied. Data show that the liver was a target organ of all pesticides and their mixtures. Increases in liver weight were observed and histopathological examination revealed centrilobular hepatocellular hypertrophy and cytoplasm degeneration for all treatment conditions. No accumulation of hepatic triglycerides was reported. Tissue residue analysis showed altered pesticide residues in the liver and the kidney when being in mixture as compared to the levels of pesticide residues for the single compound treatment, indicating possible toxicokinetic interactions. Overall, all mixtures appeared to follow the additivity concept, even though quantitative analysis was limited for some endpoints due to the semi-quantitative nature of the data, raising no specific concern for the risk assessment of the examined pesticides.

Bile acids play an active role in fat metabolism and, in high-fat diets, elevated concentrations of fecal bile acids may be related to an increased risk of colorectal cancer. This study investigated concentrations of fecal and serum bile acids in 36 vegans and 36 omnivores. The reduced rank regression was used to identify dietary patterns associated with fecal bile acids. Dietary patterns were derived with secondary and conjugated fecal bile acids as response variables and 53 food groups as predictors. Vegans had higher fiber (p &lt, 0.01) and lower fat (p = 0.0024) intake than omnivores. In serum, primary and glycine-conjugated bile acids were higher in vegans than in omnivores (p &le, 0.01). All fecal bile acids were significantly lower in vegans compared to omnivores (p &lt, 0.01). Processed meat, fried potatoes, fish, margarine, and coffee contributed most positively, whereas muesli most negatively to a dietary pattern that was directly associated with all fecal bile acids. According to the pattern, fat intake was positively and fiber intake was inversely correlated with bile acids. The findings contribute to the evidence that, in particular, animal products and fat may play a part in higher levels of fecal bile acids.

Exposure to complex chemical mixtures requires a tiered strategy for efficient mixture risk assessment. As a part of the EuroMix project we developed an adverse outcome pathway (AOP)-based assay toolbox to investigate the combined effects of the liver steatosis-inducing compounds imazalil, thiacloprid, and clothianidin in human HepaRG hepatocarcinoma cells. Compound-specific relative potency factors were determined using a benchmark dose approach. Equipotent mixtures were tested for nuclear receptor activation, gene and protein expression, and triglyceride accumulation, according to the molecular initiating events and key events proposed in the steatosis AOP. All three compounds affected the activity of nuclear receptors, but not key genes/proteins as proposed. Triglyceride accumulation was observed with three different methods. Mixture effects were in agreement with the assumption of dose additivity for all the combinations and endpoints tested. Compound-specific RPFs remained similar over the different endpoints studied downstream the AOP. Therefore, it might be possible to reduce testing to a smaller battery of key tests. The results demonstrate the suitability of our in vitro assay toolbox, integrated within an AOP framework and combined with the RPF approach, for the analysis of steatotic effects of chemical mixtures. However, mRNA results suggest that the steatosis AOP still needs improvement.

Activation of nuclear receptors (NR), for example the retinoid-X-receptors (RXR) or the liver-X-receptors (LXR), plays a crucial role as the molecular initiating event in the adverse outcome pathway for liver steatosis. The downstream biological consequences of NR interactions are still not fully understood, especially with multi-receptor-activating compounds and their mixtures. While the default assumption for mixture risk assessment is dose addition, the potential of combinations of synthetic RXR agonists to exert synergistic effects has been shown in the context of NR activation studies. The fact that RXR and LXR are heterodimerization partners raises the question whether combinations of LXR and RXR agonists may cause synergistic effects. Compounds with defined properties were chosen to examine their interactions regarding the activation of RXR and LXR, as well as the steatosis-related key events target gene activation and triglyceride accumulation, using the human HepaRG liver cell model. Synergistic effects were determined for cellular triglyceride accumulation, especially at high compound concentrations, as evaluated using five different mathematical models. Altered LXRα activation in the presence of RXR agonists was observed, and synergistic effects on LXR target genes were identified as a presumably underlying mechanism of the observed synergistic effect. These findings challenge the general validity of dose addition as the default assumption for mixture effects, and point toward the need for a mode of action-based risk assessment for chemical mixtures.