Brominated Dioxins in Egg, Broiler, and Feed Additives: Significance of Bioassay-Directed Screening for Identification of Emerging Risks in Food.

Food authorities aim to safeguard our food. This requires sensitive analyses to guarantee detection of both banned and regulated substances at low concentrations. At the same time, broad screening methods are needed to identify new emerging risks. For this purpose, effect-based bioassays combined with mass spectrometric analyses offer an advantage. During the regular monitoring of dioxins in agricultural products, a discrepancy was observed between the results of the DR CALUX (Dioxin-Responsive Chemical Activated Luciferase gene Expression) bioassay and the confirmatory gas chromatographic high resolution mass spectrometric (GC-HRMS) analysis in egg and broiler fat samples. The response in the bioassay was high, suggesting a clear exceedance of the maximum limits of dioxins in these samples, yet regulated dioxins or dl-PCBs were not detected by GC/HRMS analysis. Ultimately, a broad screening analysis using GC-HRMS resulted in the identification of 2,3,7,8-tetrabromo-dibenzofuran (2,3,7,8-TBDF) in both egg and broiler fat. To investigate the potential source of this brominated furan contaminant, different samples were analyzed: bedding material, poultry feed, feed additives (choline chloride and l-lysine), and seaweed. The poultry feed and feed additives all contained 2,3,7,8-TBDF. Using a feed-to-food transfer model, it became clear that the poultry feed was probably the source of 2,3,7,8-TBDF in broilers and eggs through a feed additive like L-lysine or choline chloride. This study underlines the importance of using a combination of effect-based screening assays with sensitive analytical methods to detect potential new and emerging risks.

Exploring the potential of using ion mobility-mass spectrometry to separate matrix interferences from analytes in food control.

During residue analysis in complex matrices for food safety purposes, interfering signals can sometimes overlap with those of the analyte of interest. Access to an additional separation dimension besides chromatographic and mass separation, such as ion mobility, can aid in removing interfering signals, allowing for correct analyte identification in these cases. In our laboratory, during routine LC-MS/MS analysis of liver samples for growth promoter residues, an interfering signal was found that matches the retention time and m/z values for stanozolol, a synthetic anabolic steroid. In the present work, the performance of a liquid chromatography coupled to ion mobility mass spectrometry (LC-IM-MS) method has been evaluated to study whether this LC-MS/MS false positive in liver samples could be eliminated by LC-IM-MS analysis. A cyclic ion mobility system already allowed the separation of stanozolol from the interfering peak after only one pass, showing a significant improvement compared to the conventional LC-MS/MS method. Additionally, collisional cross section (CCS) values were calculated and successfully compared with those from literature for identification purposes, eventually allowing both the identification and quantification of stanozolol in this complex matrix.

Occurrence and associated health risks of pyrrolizidine alkaloids in supplements marketed in Ghana for improved sexual performance.

Pyrrolizidine alkaloids (PAs) are noted for their hepatotoxic, genotoxic, and carcinogenic effects in animals and humans following metabolic activation in the liver. In this study, herbal supplements sold in Ghana for sexual improvement were analysed for the presence of 64 PAs using LC-MS/MS analysis. Up to 17 different PAs were identified in 19 out of the 37 samples analysed. The sum of PAs in samples ranged from 5 to 3204 µg kg-1. Since the PA content in the herbal medicinal preparations was generally lower than in honey samples, their presence was mainly attributed to cross-contamination. The observed levels would result in estimated daily intakes from 0.01 to 12 µg per day or 0.0002 to 0.2 µg kg-1 bw day-1 for a person weighing 70 kg. The margins of exposure ranged from 1200 to 1,400,000 with eight samples showing values below 10,000, thus indicating a health concern.

A Multiplex Gene Expression Assay for Direct Measurement of RNA Transcripts in Crude Lysates of the Nematode Caenorhabditis elegans Used as a Bioanalytical Tool.

Gene expression profiling in Caenorhabditis elegans has been demonstrated to be a potential bioanalytical tool to detect the toxic potency of environmental contaminants. The RNA transcripts of genes responding to toxic exposure can be used as biomarkers for detecting these toxins. For routine application in environmental quality monitoring, an easy-to-use multiplex assay is required to reliably quantify expression levels of these biomarkers. In the present study, a bead-based assay was developed to fingerprint gene expression in C. elegans by quantitating messenger RNAs (mRNAs) of multiple target genes directly from crude nematode lysates, circumventing RNA extraction and purification steps. The assay uses signal amplification rather than target amplification for direct measurement of toxin-induced RNA transcripts. Using a 50-gene panel, the expression changes of four candidate reference genes and 46 target mRNAs for various contaminants and wastewaters were successfully measured, and the expression profiles indicated the type of toxin present. Moreover, the multiplex assay response was in line with previous results obtained with more time-consuming reverse-transcription quantitative polymerase chain reaction and microarray analyses. In addition, the transcriptomic profiles of nematodes exposed to wastewater samples and extracts prepared from tissues of swimming crabs were evaluated. The profiles indicated the presence of organic pollutants. The present study illustrates the successful development of a multiplex fluorescent bead-based approach using nematode C. elegans crude lysates for gene expression profiling of target RNAs. This method can be used to routinely fingerprint the presence of toxic contaminants in environmental samples and to identify the most biologically active fraction of the contaminant mixture in a toxicity identification and evaluation approach. Environ Toxicol Chem 2023;42:130-142. © 2022 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Presence and risks of polycyclic aromatic hydrocarbons, dioxins and dioxin-like PCBs in dietary plant supplements as elucidated by a combined DR CALUX® bioassay and GC-HRMS based approach.

Plant-based dietary supplements may contain undesirable contaminants such as polycyclic aromatic hydrocarbons, dioxins and dioxin-like polychlorinated biphenyls (dl-PCBs) due to the sources of raw materials or processing methods used. The presence of these contaminants in a series of herbal supplements sold on the Ghanaian market for improving sexual performance was examined using the DR CALUX® bioassay in combination with GC-HRMS analysis. Overall, cell responses at 4 and 48 h exposure to extracts prepared without an acid-silica clean-up were relatively higher than the responses obtained from extracts prepared with an acid-silica clean-up. This indicated that the 40 supplements contained only low levels of stable aryl hydrocarbon receptor (AhR) agonists like polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs) and dl-PCBs, while some contained substantial amounts of less stable AhR-agonists. Ten supplements selected for confirmation with GC-HRMS analysis contained PCDD/Fs and dl-PCBs at levels ranging from 0.01 to 0.19 pg toxic equivalent (TEQ)/g only, while the level of the sum of 4 polycyclic aromatic hydrocarbons (Σ4PAHs) representing less stable AhR agonists, ranged from not detected (ND) to 25.5 ng/g. These concentrations were in line with the responses observed in the DR CALUX® bioassay. The concentration of PCDD/Fs and dl-PCBs corresponded to estimated daily intakes (EDIs) ranging from 0.01 to 1.20 pg TEQ/day, or 0.001 to 0.12 pg TEQ/kg bw/week for a 70 kg bw consumer, which was below the established tolerable weekly intake (TWI) of 2 pg TEQ/kg bw/week, thus indicating low concern for consumers' health. Similarly, the EDIs based on the detected Σ4PAHs in supplements ranged from 7.2 to 111 ng/day, or 0.1 to 1.6 ng/kg bw/day, which corresponded to MOE values above 10,000, indicating a low health concern.

Identification of phosphodiesterase type-5 (PDE-5) inhibitors in herbal supplements using a tiered approach and associated consumer risk.

The use of herbal supplements for improved sexual performance is a common practice amongst the youth and some senior citizens in Ghana. These products are considered 'natural' and greatly preferred over synthetic alternatives due to the assurance of little to no adverse effects by producers. However, the high rate of adulteration often compromises their safety. Forty herbal supplements, of which 25 were previously shown to result in medium to high intake of phosphodiesterase type-5 (PDE-5) inhibitors using a PDE-Glo bioassay, were further investigated using liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis to examine the reliability of the bioassay and whether the observed higher responses could be ascribed to inherent plant constituents or adulterants. Results showed significant amounts of vardenafil, tadalafil and especially sildenafil, in 2, 1 and 10 samples, respectively, with total concentration levels resulting in estimated daily intakes (EDIs) above 25 mg sildenafil equivalents with six supplements even having EDIs above 100 mg sildenafil equivalents. Only one sample contained a natural ingredient (icariin), but its concentration (0.013 mg g-1) was too low to explain the observed potency in the bioassay. The estimated concentrations of PDE-5 inhibitors in 35 supplements, according to the bioassay, were in line with those of the LC-MS/MS analysis. However, discrepancies were observed for five supplements. Further examination of one of the latter supplements using the PDE-Glo bioassay to select the positive fraction and further examination with LC-MS/MS and 1H-NMR revealed the presence of hydroxythiohomosildenafil, a sildenafil analogue not yet included in the liquid chromatography-mass spectrometry reference library. This study demonstrates the significance of applying a tiered approach, where the use of a bioassay is followed by chemical analysis of bioactive samples in order to identify unknown bioactive compounds.

Microsphere Peptide-Based Immunoassay for the Detection of Recombinant Bovine Somatotropin in Injection Preparations.

The use of peptides in immunoassays can be favored over the use of the full protein when more cost effective or less toxic approaches are needed, or when access to the full protein is lacking. Due to restricted access to recombinant bovine somatotropin (rbST), a protein enhancing growth and lactating performances of livestock, which use has been banned in the EU, Canada and Australia (amongst others), we developed a peptide-based biorecognition assay on an imaging planar array analyzer. For this, we identified the rbST epitope that is responsible for binding to the rbST-targeting monoclonal antibody 4H12 (MAb 4H12) to be 115DLEEGILALMR125. This linear peptide was synthesized and coupled to microspheres, after which it was tested in a biorecognition competitive inhibition assay format. We observed IC50 values of approximately 0.11 µg mL-1, which are lower than observed for the full rbST protein (IC50 = 0.20 µg mL-1). Importantly, there was no binding with the scrambled peptide. Preliminary results of directly coupled peptides in a microsphere biorecognition assay for detection of rbST are presented. Real-life applicability for detection of somatotropins (STs) in injection preparations of bovine-, porcine- and equine ST are shown. This newly developed immunoassay strongly supports future developments of peptide-based immunoassays to circumvent the limited access to the full protein.

Characterisation and validation of an in vitro transactivation assay based on the 22Rv1/MMTV_GR-KO cell line to detect human androgen receptor agonists and antagonists.

We describe the characterisation and validation of an androgen receptor (AR) transactivation assay for detection of AR agonists and antagonists using a stably transfected human prostate cancer cell line. This 22Rv1/mouse mammary tumour virus glucocorticoid knock-out cell line based AR transactivation assay was validated by criteria in Organisation for Economic Cooperation and Development Guidance Document 34 to determine if the assay performed equally well to the AR EcoScreen Assay included in Test Guideline for AR Transactivation (OECD TG 458). There was no Glucocorticoid Receptor (GR) crosstalk, and no changes in the AR DNA sequence in cells after the successful knock out of GR. Subsequently, the concordance of classifications of the 22 test chemicals was 100% in all laboratories. The AR agonistic and antagonistic inter-laboratory coefficients of variation based on log[10% effect for 10 nM DHT, PC10] and log[inhibitory response of 800 pM DHT by at 30%, IC30] from comprehensive tests were 2.75% and 2.44%, respectively. The AR agonist/antagonist test chemical classifications were consistent across AR EcoScreen ARTA assay data for 82/89%, and the balanced accuracy, sensitivity, and specificity were 83/90%, 88/100% and 78/80%, respectively. This assay was successfully validated and was approved for inclusion in TG 458 in 2020.

PDE-5 inhibitors in selected herbal supplements from the Ghanaian market for better erectile function as tested by a bioassay.

Herbal supplements sold as 'all natural' on various markets in Accra (Ghana) and advertised as highly efficacious in treating erectile dysfunction (ED) were bought and analysed by a PDE-5 enzyme inhibition assay. The claimed efficacy of these products could be the result of inherent plant constituents, but also of intentionally added pharmaceuticals. Medically, ED is treated with potent inhibitors of the phosphodiesterase-5 (PDE-5) enzyme, as in the case of sildenafil. To test the efficacy of the Ghanaian supplements, extracts were made and tested using a PDE-Glo phosphodiesterase assay, a luminescent high-throughput screening (HTS) method. Results revealed that about 90% of the selected samples were able to inhibit PDE-5 activity to a high extent. Estimated concentrations in sildenafil equivalents ranged from traces to very high, with 25 samples (62.5%) pointing at daily doses higher than 25 mg sildenafil equivalents and 9 (22.5%) of these at doses higher than the maximal recommended daily intake of 100 mg sildenafil equivalents. Further investigations are needed to confirm if the observed effects are due to inherent plant constituents or merely the result of added synthetic PDE-5 enzyme inhibitors, especially because doses above 100 mg sildenafil equivalents per day may result in severe health risks.

Detection of methionine- and alanine-recombinant bovine somatotropins and their induced antibodies in serum and milk of cows suggests blood-milk barrier specificity for these compounds.

The elimination of recombinant bovine somatotropin (rbST) and its induced antibodies through milk of 2 formulations is studied to propose a control strategy for its use or abuse. Two dairy cows were treated with alanine-rbST (Ala-rbST), which is identical to endogenous bovine somatotropin, and ten dairy cows were treated with methionine-rbST (Met-rbST), which differs by 1 amino acid from endogenous bovine somatotropin. We developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method able to measure rbST at a decision limit (CCα) of 0.8 ng/mL and 2.3 ng/mL for serum and milk, respectively. The results show that the administered Ala-rbST is transferred from blood to milk but that this is not the case for Met-rbST. This suggests a blood-milk barrier-related specificity for these compounds. In addition, rbST-induced antibodies were formed in animals treated with Ala-rbST and those treated with Met-rbST. In both treatments, the rbST-induced antibodies were transferred from blood to milk, showing no blood-milk barrier specificity for these antibodies. These elimination patterns show that, for enforcement purposes, the detection of rbST-induced antibodies in tank milk can serve to screen for rbST administration, and subsequent confirmatory serum analysis by LC-MS/MS is needed to identify whether Ala-rbST or Met-rbST has been used.

Impurities in technical mixtures of chlorinated paraffins show AhR agonist properties as determined by the DR-CALUX bioassay.

Chlorinated paraffins (CPs) are produced at more than one million tons per year. Technical CPs mixtures may contain impurities, which end up in consumer products. In the present study, 17 technical CPs mixtures were investigated for the potential occurrence of potential impurities. By applying the DR-CALUX bioassay, 3 out of 17 technical mixtures were shown to elicit responses at 4 h exposure time, but much lower at 48 h. Constitutional defined CPs materials did not show responses. Subsequently different groups of known AhR-agonists and compounds suspected to be present in technical CPs mixtures were investigated. Benzene, (poly)chlorobenzene, non-dioxin like polychlorinated naphthalenes (PCNs), and three-ringed polyaromatic hydrocarbons (PAHs) did not result in a significant response at 4 h or 48 h. TCDD, non-ortho PCBs, dioxin-like PCNs, four or five ringed PAHs and their chlorinated analogues resulted in a significant response. TCDD and the non-ortho PCBs showed the highest potency and stability, while dioxin-like PCNs, PAHs, and the chlorinated PAHs were clearly inactivated (metabolized) at longer incubation. Altogether, the present findings substantiate that AhR-mediated responses of CPs technical mixtures in the DR-CALUX bioassay are caused by impurities, most likely some intermediate stable AhR-agonists such as dioxin-like PCNs or (chlorinated) PAHs. The current study shows that impurities in CPs technical mixtures need to be investigated for assessing the safety of technical CPs mixtures.

Quantitative in vitro-to-in vivo extrapolation (QIVIVE) of estrogenic and anti-androgenic potencies of BPA and BADGE analogues.

The goal of the present study was to obtain an in vivo relevant prioritization method for the endocrine potencies of different polycarbonate monomers, by combining in vitro bioassay data with physiologically based kinetic (PBK) modelling. PBK models were developed for a selection of monomers, including bisphenol A (BPA), two bisphenol F (BPF) isomers and four different bisphenol A diglycidyl ethers (BADGEs), using in vitro input data. With these models, the plasma concentrations of the compounds were simulated, providing means to estimate the dose levels at which the in vitro endocrine effect concentrations are reached. The results revealed that, whereas the in vitro relative potencies of different BADGEs (predominantly anti-androgenic effects) can be up to fourfold higher than BPA, the estimated in vivo potencies based on the oral equivalent doses are one to two orders of magnitude lower than BPA because of fast detoxification of the BADGEs. In contrast, the relative potencies of 2,2-BPF and 4,4-BPF increase when accounting for the in vivo availability. 4,4-BPF is estimated to be fivefold more potent than BPA in humans in vivo in inducing estrogenic effects and both 2,2-BPF and 4,4-BPF are estimated to be, respectively, 7 and 11-fold more potent in inducing anti-androgenic effects. These relative potencies were considered to be first-tier estimates, particularly given that the potential influence of intestinal metabolism on the in vivo availability was not accounted for. Overall, it can be concluded that both 2,2-BPF and 4,4-BPF are priority compounds.

Detection and profiling of diarrheic marine biotoxins in shellfish by mRNA analysis of exposed Caco-2 cells using qRT-PCR and multiplex magnetic bead-based assays.

The mouse bioassay for the detection of marine biotoxins in shellfish products is 40 years old and still in use. A full ban or total replacement of this in vivo test has been postponed because of the fear that current chemical-based detection methods could miss a new emerging toxin. In order to fully replace the mouse bioassay, more efforts are needed on the search for functional assays with specific endpoints. Gene expression elicited by diarrheic shellfish poisons in Caco-2 cells allowed us to determine three 'DSP profiles', i.e. OA/DTX, AZA-YTX and PTX profiles. Twelve marker genes were selected to envision the three profiles. qRT-PCR is relatively cheap and easy, and although its multiplex capacity is limited to 5 genes, this turned out to be sufficient to show the three expected profiles. The use of the multiplex magnetic bead-based assay turned out to be even a slightly better alternative, allowing the use of all twelve selected marker genes and 2 reference genes, and resulting in clear profiles with for some genes even higher induction factors as obtained by qRT-PCR. When analysing blank and contaminated shellfish samples with this multiplex magnetic bead-based assay, the contaminated samples could easily be distinguished from the blank samples, showing the expected profiles. This work is one step further on the final replacement of the mouse bioassay, e.g. by combining the neuro-2a bioassay for screening and detection with analytical chemical analyses and the multiplex magnetic bead-based assay for confirmation of known and unknown toxins respectively.

A Strategy to Replace the Mouse Bioassay for Detecting and Identifying Lipophilic Marine Biotoxins by Combining the Neuro-2a Bioassay and LC-MS/MS Analysis.

Marine biotoxins in fish and shellfish can cause several symptoms in consumers, such as diarrhea, amnesia, or even death by paralysis. Monitoring programs are in place for testing shellfish on a regular basis. In some countries testing is performed using the so-called mouse bioassay, an assay that faces ethical concerns not only because of animal distress, but also because it lacks specificity and results in high amounts of false positives. In Europe, for lipophilic marine biotoxins (LMBs), a chemical analytical method using LC-MS/MS was developed as an alternative and is now the reference method. However, safety is often questioned when relying solely on such a method, and as a result, the mouse bioassay might still be used. In this study the use of a cell-based assay for screening, i.e., the neuro-2a assay, in combination with the official LC-MS/MS method was investigated as a new alternative strategy for the detection and quantification of LMBs. To this end, samples that had been tested previously with the mouse bioassay were analyzed in the neuro-2a bioassay and the LC-MS/MS method. The neuro-2a bioassay was able to detect all LMBs at the regulatory levels and all samples that tested positive in the mouse bioassay were also suspect in the neuro-2a bioassay. In most cases, these samples contained toxin levels (yessotoxins) that explain the outcome of the bioassay but did not exceed the established maximum permitted levels.

Screening for the presence of lipophilic marine biotoxins in shellfish samples using the neuro-2a bioassay.

The neuro-2a bioassay is considered as one of the most promising cell-based in vitro bioassays for the broad screening of seafood products for the presence of marine biotoxins. The neuro-2a assay has been shown to detect a wide array of toxins like paralytic shellfish poisons (PSPs), ciguatoxins, and also lipophilic marine biotoxins (LMBs). However, the neuro-2a assay is rarely used for routine testing of samples due to matrix effects that, for example, lead to false positives when testing for LMBs. As a result there are only limited data on validation and evaluation of its performance on real samples. In the present study, the standard extraction procedure for LMBs was adjusted by introducing an additional clean-up step with n-hexane. Recovery losses due to this extra step were less than 10%. This wash step was a crucial addition in order to eliminate false-positive outcomes due to matrix effects. Next, the applicability of this assay was assessed by testing a broad range of shellfish samples contaminated with various LMBs, including diarrhetic shellfish toxins/poisons (DSPs). For comparison, the samples were also analysed by LC-MS/MS. Standards of all regulated LMBs were tested, including analogues of some of these toxins. The neuro-2a cells showed good sensitivity towards all compounds. Extracts of 87 samples, both blank and contaminated with various toxins, were tested. The neuro-2a outcomes were in line with those of LC-MS/MS analysis and support the applicability of this assay for the screening of samples for LMBs. However, for use in a daily routine setting, the test might be further improved and we discuss several recommended modifications which should be considered before a full validation is carried out.

Whole genome mRNA transcriptomics analysis reveals different modes of action of the diarrheic shellfish poisons okadaic acid and dinophysis toxin-1 versus azaspiracid-1 in Caco-2 cells.

A study with DNA microarrays was performed to investigate the effects of two diarrhetic and one azaspiracid shellfish poison, okadaic acid (OA), dinophysistoxin-1 (DTX-1) and azaspiracid-1 (AZA-1) respectively, on the whole-genome mRNA expression of undifferentiated intestinal Caco-2 cells. Previously, the most responding genes were used to develop a dedicated array tube test to screen shellfish samples on the presence of these toxins. In the present study the whole genome mRNA expression was analyzed in order to reveal modes of action and obtain hints on potential biomarkers suitable to be used in alternative bioassays. Effects on key genes in the most affected pathways and processes were confirmed by qPCR. OA and DTX-1 induced almost identical effects on mRNA expression, which strongly indicates that OA and DTX-1induce similar toxic effects. Biological interpretation of the microarray data indicates that both compounds induce hypoxia related pathways/processes, the unfolded protein response (UPR) and endoplasmic reticulum (ER) stress. The gene expression profile of AZA-1 is different and shows increased mRNA expression of genes involved in cholesterol synthesis and glycolysis, suggesting a different mode of action for this toxin. Future studies should reveal whether identified pathways provide suitable biomarkers for rapid detection of DSPs in shellfish.

Effects and detection of Nandrosol and ractopamine administration in veal calves.

The present study describes different effects of the selective androgen receptor modulator (SARM) nandrolone phenylpropionate (Nandrosol) and the ß-agonist ractopamine administration in veal calves, and it investigates different strategies applied to trace these molecules. Morphological changes of gonads and accessory glands attributed to androgen effects, such as testicular atrophy, seminiferous tubule diameter reduction and hyperplasia of prostate epithelium, were detected, although SARMs are not described to cause these lesions. The gene expression analysis showed an anabolic activity of Nandrosol in Longissimus dorsi muscle, where myosin heavy chain (MYH) was significantly up-regulated. An IGF1 increase was weakly significant only in Vastus lateralis muscle. In conclusion, the anatomo-histopathological observations and the MYH mRNA up-regulation in Longissimus dorsi muscle confirm the androgenic treatment in experimental animals. The biosensor assay was not enough sensitive to detect residues in urines and only the direct chemical analysis of urine samples confirmed both ß-agonist and SARM treatment.

Effects of Digested Onion Extracts on Intestinal Gene Expression: An Interspecies Comparison Using Different Intestine Models.

Human intestinal tissue samples are barely accessible to study potential health benefits of nutritional compounds. Numbers of animals used in animal trials, however, need to be minimalized. Therefore, we explored the applicability of in vitro (human Caco-2 cells) and ex vivo intestine models (rat precision cut intestine slices and the pig in-situ small intestinal segment perfusion (SISP) technique) to study the effect of food compounds. In vitro digested yellow (YOd) and white onion extracts (WOd) were used as model food compounds and transcriptomics was applied to obtain more insight into which extent mode of actions depend on the model. The three intestine models shared 9,140 genes which were used to compare the responses to digested onions between the models. Unsupervised clustering analysis showed that genes up- or down-regulated by WOd in human Caco-2 cells and rat intestine slices were similarly regulated by YOd, indicating comparable modes of action for the two onion species. Highly variable responses to onion were found in the pig SISP model. By focussing only on genes with significant differential expression, in combination with a fold change > 1.5, 15 genes showed similar onion-induced expression in human Caco-2 cells and rat intestine slices and 2 overlapping genes were found between the human Caco-2 and pig SISP model. Pathway analyses revealed that mainly processes related to oxidative stress, and especially the Keap1-Nrf2 pathway, were affected by onions in all three models. Our data fit with previous in vivo studies showing that the beneficial effects of onions are mostly linked to their antioxidant properties. Taken together, our data indicate that each of the in vitro and ex vivo intestine models used in this study, taking into account their limitations, can be used to determine modes of action of nutritional compounds and can thereby reduce the number of animals used in conventional nutritional intervention studies.

Dietary supplement for energy and reduced appetite containing the ß-agonist isopropyloctopamine leads to heart problems and hospitalisations.

In 2013 the Dutch authorities issued a warning against a dietary supplement that was linked to 11 reported adverse reactions, including heart problems and in one case even a cardiac arrest. In the UK a 20-year-old woman, said to have overdosed on this supplement, died. Since according to the label the product was a herbal mixture, initial LC-MS/MS analysis focused on the detection of plant toxins. Yohimbe alkaloids, which are not allowed to be present in herbal preparations according to Dutch legislation, were found at relatively high levels (400-900 mg kg(-1)). However, their presence did not explain the adverse health effects reported. Based on these effects the supplement was screened for the presence of a ß-agonist, using three different biosensor assays, i.e. the validated competitive radioligand ß2-adrenergic receptor binding assay, a validated ß-agonists ELISA and a newly developed multiplex microsphere (bead)-based ß-agonist assay with imaging detection (MAGPIX(®)). The high responses obtained in these three biosensors suggested strongly the presence of a ß-agonist. Inspection of the label indicated the presence of N-isopropyloctopamine. A pure standard of this compound was bought and shown to have a strong activity in the three biosensor assays. Analysis by LC-full-scan high-resolution MS confirmed the presence of this 'unknown known' ß3-agonist N-isopropyloctopamine, reported to lead to heart problems at high doses. A confirmatory quantitative analysis revealed that one dose of the preparation resulted in an intake of 40-60 mg, which is within the therapeutic range of this compound. The case shows the strength of combining bioassays with chemical analytical techniques for identification of illegal pharmacologically active substances in food supplements.

Estrogen Receptor Agonists and Antagonists in the Yeast Estrogen Bioassay.

Cell-based bioassays can be used to predict the eventual biological activity of a substance on a living organism. In vitro reporter gene bioassays are based on recombinant vertebrate cell lines or yeast strains and especially the latter are easy-to-handle, cheap, and fast. Moreover, yeast cells do not express estrogen, androgen, progesterone or glucocorticoid receptors, and are thus powerful tools in the development of specific reporter gene systems that are devoid of crosstalk from other hormone pathways. This chapter describes our experience with an in-house developed RIKILT yeast estrogen bioassay for testing estrogen receptor agonists and antagonists, focusing on the applicability of the latter.