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.

Dose addition in mixtures of compounds with dissimilar endocrine modes of action in in vitro receptor activation assays and the zebrafish sexual development test.

BACKGROUND: Human exposure to pesticides is being associated with feminisation for which a decrease of the anogenital distance (AGD) is a sensitive endpoint. Dose addition for the cumulative risk assessment of pesticides in food is considered sufficiently conservative for combinations of compounds with both similar and dissimilar modes of action (MoA). OBJECTIVE: The present study was designed to test the dose addition hypothesis in a binary mixture of endocrine active compounds with a dissimilar mode of action for the endpoint feminisation. METHODS: Compounds were selected from a list of chemicals of which exposure is related to a decrease of the AGD in rats and completed with reference compounds. These chemicals were characterised using specific in vitro transcriptional activation (TA) assays for estrogenic and androgenic properties, leading to a final selection of dienestrol as an ER-agonist and flutamide, linuron, and deltamethrin as AR-antagonists. These compounds were then tested in an in vivo model, i.e. in zebrafish (Danio rerio), using sex ratio in the population as an endpoint in order to confirm their feminising effect and MoA. Ultimately, the fish model was used to test a binary mixture of flutamide and dienestrol. RESULTS: Statistical analysis of the binary mixture of flutamide and dienestrol in the fish sexual development tests (FSDT) with zebrafish supported dose addition.

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.

Neonicotinoid Microsphere Immunosensing for Profiling Applications in Honeybees and Bee-Related Matrices.

Neonicotinoids are the most commonly used insecticides due to their effectiveness. However, non-targeted insects, especially bees, are also affected by neonicotinoids. Therefore, neonicotinoid application can contribute to the declining bee populations worldwide. The presented study describes the development of novel competitive, fluorescent microsphere-based suspension immunoassays for neonicotinoid profiling and their application to bees and essential bee-related matrices, using the Multi-Analyte Profiling (xMAP) technology. For the construction of these neonicotinoid microsphere immunoassays (nMIAs), neonicotinoid-ovalbumin conjugates were coupled to unique fluorescent, paramagnetic microspheres, which competed with the free neonicotinoids that were present in test samples for interacting with the corresponding, specific antibodies. In total, five independent nMIA's were developed for the detection of imidacloprid, acetamiprid, clothianidin, thiacloprid, thiamethoxam, dinotefuran, nitenpyram and imidaclothiz with the limits of detection being for 0.01 ng/mL, 0.01 ng/mL, 0.02 ng/mL, 0.02 ng/mL, 0.003 ng/mL, 2.95 ng/mL, 0.09 ng/mL and 0.04 ng/mL, respectively. The developed nMIAs were applied to fortified matrices including surface water, pollen, honey and honeybees. All of the neonicotinoids, except dinotefuran, could be sensitively detected in all of the tested environmental matrices and bees, with there being sensitivities of 1 ng/mL in water and 10 ng/g in solid materials. These nMIAs provide a rapid profiling method for all of the common neonicotinoids, including those that are banned by the European Union for outdoor use. The developed method can contribute to healthy and sustainable beekeeping, globally, via its application in the apiary environment.

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.

Maternal exposure to mixtures of dienestrol, linuron and flutamide. Part II: Endocrine-related gene expression assessment on male offspring rat testes.

Exposure to endocrine-disrupting compounds (EDCs) during pregnancy and early development can lead to adverse developmental outcomes in offspring. One of the endpoints of concern is feminization. The present study aimed to investigate for any possible correlations with endocrine sensitive parameters in the testes of male rat offspring following dam exposure to three EDCs by assessing the expression of endocrine-related genes. Dienestrol (DIES) [0.37-6.25 µg/kg bw/day], linuron (LIN) [1.5-50 mg/kg bw/day], flutamide (FLU) [3.5-50 mg/kg bw/day] as well as their binary mixtures were administered to sexually mature female rats from gestation day (GD) 6 until postnatal day (PND) 21. Gene expression analysis of Star, Cyp11a1, Cyp17a1, Hsd3b2, Pgr and Insl3 was performed by RT-qPCR. Administration of the anti-androgen FLU alone significantly upregulated Cyp11a1 and Cyp17a1 gene expression while administration of LIN and DIES alone did not alter significantly gene expression. The effects of the binary mixtures on gene expression were not as marked as those seen after single compound administrations. Deregulation of Cyp17a1 in rat pup testis, following administration of FLU alone or in mixtures to dams, was significantly correlated with the observed feminization endpoints in male pups.

Maternal exposure to mixtures of dienestrol, linuron and flutamide. Part I: Feminization effects on male rat offspring.

Exposure to endocrine-disrupting compounds (EDCs) during pregnancy can result in negative health effects in later generations, including sex changes and feminization. The present study assessed the feminization effects on male offspring rats of three EDCs: Dienestrol (DIES), Linuron (LIN), and Flutamide (FLU). Sexually mature female rats were exposed from gestation day (GD) 6 until postnatal day (PND) 21 to: 0.37, 0.75, 1.5, 3.12 or 6.25 µg/kg/day of DIES, 1.5, 3, 6, 12.5, 25 or 50 mg/kg/day of LIN, 3.5, 6.7, 12.5, 25 or 50 mg/kg/day of FLU, and the following mixtures: FLU + DIES (mg/kg/day+µg/kg/day), 3.5 + 0.37, or 3.5 + 3, 25 + 0.37, or 25 + 3; FLU + LIN (mg/kg/day + mg/kg/day), 3.5 + 12.5, or 25 + 12.5; and DIES + LIN (µg/kg/day + mg/kg/day), 0.37 + 12.5, or 3 + 12.5. Anogenital distance (AGD), nipple retention (NR) and cryptorchidism were evaluated. FLU produced a decrease of AGD, an increase of NR, and an increase of cryptorchidism at the highest dose. None of these three endpoints were significantly affected by LIN or DIES treatments alone. Combinations of FLU + LIN and FLU + DIES increased NR, and decreased AGD, while DIES + LIN did not produce any effects in male pups. Results show that FLU is able to induce feminization in male pups, while binary combinations of LIN and DIES did not modify the effects produced by FLU.

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.

BPA, BADGE and analogues: A new multi-analyte LC-ESI-MS/MS method for their determination and their in vitro (anti)estrogenic and (anti)androgenic properties.

Information on the occurrence and endocrine potencies of analogues of bisphenol A (BPA) and diglycidyl ester derivatives (BDGEs) of BPA and BPF is limited. Such information is, however, important as the current debate on BPA and the lowered BPA migration limit in Europe may provide an incentive for application of structural analogues. A new sensitive multi-analyte LC-ESI-MS/MS method was developed to measure 17 bisphenols (BPs) and 6 BDGEs in food, beverages and drinkware. Yeast based bioassays were used to determine the in vitro (anti)estrogenic and (anti)androgenic properties of these and 7 additional BPs and BDGEs. Drinkware of polycarbonate and other materials were analysed for BPs and BDGEs. Only BPA and BPS and both at trace levels were found in a few containers. A limited number of (canned) foods and beverages were also analysed. BPA was the most frequently detected BP (ranged from 0.03 ng mL-1 in a beverage sample to 68 ng g-1 in food). Other BPs detected were BPS, 2,2-BPF and 4,4-BPF. In addition BADGE, BADGE.HCl, BADGE.H2O and BADGE.2H2O were detected from 0.08 ng mL-1 in a beverage sample to 3.3 ng g-1 in food. In vitro testing showed that most BPs exhibited an equal or higher estrogenic potency than BPA and most of them also showed a higher anti-androgenic potency, i.e. BPB, BPCl, BPC, BPE, 4,4-BPF, BPP, BPAF, and BPTMC. Some BPs and BDGEs were not estrogenic, but showed an anti-estrogenic effect and were anti-androgenic too. BPS was only weakly estrogenic and BADGE.2H2O and BFDGE.2H2O showed no in vitro activity. The present data show that in addition to BPA, other BPs and BDGEs can be present in food and drinks, some displaying in vitro endocrine activities.

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.

First Report on the Occurrence of Tetrodotoxins in Bivalve Mollusks in The Netherlands.

Tetrodotoxin (TTX) is traditionally associated with seafood from tropical regions, but recently TTX was detected in bivalve mollusks in more temperate European waters. In The Netherlands it was therefore decided to monitor TTX in shellfish harvested from Dutch production areas. All shellfish production areas were monitored in 2015, 2016 and 2017. Samples were analyzed using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). In total 1063 samples were investigated, and the highest concentrations were observed in 2016, i.e., 253 µg TTX/kg in oysters and 101 µg TTX/kg in mussels. No TTX analogues, with the exception of 4-epi-TTX in one single sample, were found and contaminated samples also showed positive results in the neuro-2a bioassay. The occurrence of TTX seems to be consistent over the last three years with the highest concentrations observed annually in late June. The causative organism and the reasons why specific Dutch production areas are affected while others are not, are still unclear. Initially in The Netherlands an action limit of 20 µg TTX/kg was used to ensure the safety of consumers (2016), but recently The European Food Safety Authority (EFSA) established an acute reference dose, and based on a high portion size of consuming 400 g mussels, this dose was translated into a safe concentration of 44 µg TTX per kg for shellfish. This concentration is now used as an action limit and TTX is formally included in the Dutch shellfish monitoring program.