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Exposure to PFASs is associated to several adverse health effects, such as immunotoxicity. Immunotoxic effects of PFOA and PFOS, including a reduced antibody response in both experimental animals and humans, have been reported. However, there is limited understanding of the underlying mechanisms involved. Moreover, there is only a restricted amount of immunotoxicity data available for a limited number of PFASs. In the current study the effects of 15 PFASs, including short- and long-chain perfluorinated carboxylic and sulfonic acids, fluorotelomer alcohols, and perfluoralkyl ether carboxylic acids were studied on the expression of recombinant activating gene 1 (RAG1) and RAG2 in the Namalwa human B lymphoma cell line, and on the human IL-2 promotor activity in Jurkat T-cells. Concentration-response data were subsequently used to derive in vitro relative potencies through benchmark dose analysis. In vitro relative potency factors (RPFs) were obtained for 6 and 9 PFASs based on their effect on RAG1 and RAG2 gene expression in Namalwa B-cells, respectively, and for 10 PFASs based on their inhibitory effect on IL-2 promotor activity in Jurkat T-cells. The most potent substances were HFPO-TA for the reduction of RAG1 and RAG2 gene expression in Namalwa cells (RPFs of 2.1 and 2.3 respectively), and PFDA on IL-2 promoter activity (RPF of 9.1). RAG1 and RAG2 play a crucial role in V (D)J gene recombination, a process for acquiring a varied array of antibodies crucial for antigen recognition. Hence, the effects observed in Namalwa cells might indicate a PFAS-induced impairment of generating a diverse range of B-cells essential for antigen recognition. The observed outcomes in the Jurkat T-cells suggest a possible PFAS-induced reduction of T-cell activation, which may contribute to a decline in the T-cell dependent antibody response. Altogether, the present study provides potential mechanistic insights into the reported PFAS-induced decreased antibody response. Additionally, t

Black soldier fly larvae (Hermetia illucens) receive growing interest as a potential alternative animal feed source. These insects may be exposed to insecticide residues in the rearing substrate. This study aimed to investigate the effects of six different pyrethroid and organophosphate insecticides on this insect species’ performance. The toxicity of two “model” substances for each of these classes (cypermethrin; pirimiphos-methyl) was quantified, with and without the synergist piperonyl butoxide (PBO). Critical effect doses corresponding to 10% yield (CED10) for cypermethrin (0.4 mg/kg) and pirimiphos-methyl (4.8 mg/kg) were determined. The addition of PBO to cypermethrin enhanced its relative potency with a factor 2.6. These data were compared against the relative toxicity of two analogue substances in each class (permethrin, deltamethrin; chlorpyrifos-methyl, malathion). Results suggest that exposure to concentrations complying with legal limits can cause significant reductions in yield. Exposure to multiple substances at lower concentrations resulted in negative additive and synergistic effects. Of the tested substances, deltamethrin was most toxic, causing 94% yield at 0.5 mg/kg. Analytical results suggested that transfer of tested substances to the larval biomass was substance- and concentration-specific, but appeared to be correlated to reduced yields and the presence of PBO. Transfer of organophosphates was overall low (<2%), but ranged from 8% to 75% for pyrethroids. Due to very low limits in insect biomass (∼0.01 mg/kg), high transfer may result in noncompliance. It is recommended that rearing companies implement lower contractual thresholds, and that policymakers consider adjusting legally allowed maximum residue levels in insect feed.

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.

Per: and polyfluoroalkyl substances (PFASs) have been associated with increased blood lipids in humans. Perfluorooctanoic acid (PFOA) has been also linked with elevated alanine transferase (ALT) serum levels in humans, and in rodents the liver is a main target organ for many PFASs. With the focus on New Approach Methodologies, the chronic oral equivalent effect doses were calculated for PFOA, PFNA (perfluorononanoic acid), PFHxS (perfluorohexanesulfonic acid) and PFOS (perfluorooctane sulfonic acid) based on in vitro effects measured in the HepaRG cell line. Selected in vitro readouts were considered biomarkers for lipid disturbances and hepatotoxicity. Concentration-response data obtained from HepaRG cells on triglyceride (TG) accumulation and expression changes of 12 selected genes (some involved in cholesterol homeostasis) were converted into corresponding human dose-response data, using physiologically based kinetic (PBK) model-facilitated reverse dosimetry. Next to this, the biokinetics of the chemicals were studied in the cell system. The current European dietary PFASs exposure overlaps with the calculated oral equivalent effect doses, indicating that the latter may lead to interference with hepatic gene expression and lipid metabolism. These findings illustrate an in vitro-in silico methodology, which can be applied for more PFASs, to select those that should be prioritized for further hazard characterization.

Per- and polyfluoroalkyl substances (PFASs) are omnipresent and have been shown to induce a wide range of adverse effects, including hepatotoxicity, developmental toxicity and immunotoxicity. So far, little information is available about the mechanisms underlying the toxicity of PFASs, including those related to their immunotoxicity. Reported immunotoxic effects of PFASs include decreased antibody responses in experimental animals and humans, indicating that PFASs may, among others, affect B cell function. In the present study, we first assessed the effects of PFOA on the transcriptome of the human Namalwa B cell line using RNA seq analysis. Gene expression changes, analyzed using Ingenuity Pathway Analysis, pointed to various cellular processes affected by PFOA, including ‘B cell development’ and ‘Primary immunodeficiency signaling’. Interestingly, PFOA decreased the expression of RAG1 and RAG2, genes involved in immunoglobulin and T cell receptor V(D)J recombination. As a next step, time- and concentration-dependent changes in the expression of RAG1 and RAG2 upon exposure to PFOA, PFNA, PFHxS and PFOS were studied through RT-qPCR analysis. Analysis with the concentration–response modeling software PROAST resulted in the following potency ranking: PFNA > PFOA > PFOS > PFHxS. Altogether, the present in vitro study provides insights into the effects of selected PFASs on B cells, identifying RAG1 and RAG2 expression as possible relevant targets that may play a role in the immunotoxicity of PFASs.

In the original publication of the article, the dilution of DAPT in sentence “To promote intestinal differentiation,…” was published with an error. The “5 mM DAPT” should be “5 μM DAPT”. The original article has been updated with the correct dilution.

In vitro models are widely used to study the biotransformation of xenobiotics and to provide input parameters to physiologically based kinetic models required to predict the kinetic behavior in vivo. For farm animals this is not common practice yet. The use of slaughterhouse-derived tissue material may provide opportunities to study biotransformation reactions in farm animals. The goal of the present study was to explore the potential of slaughterhouse-derived bovine liver S9 (S9) and precision cut liver slices (PCLSs) to capture observed biotransformation reactions of lidocaine in cows. The in vitro data obtained with both S9 and PCLSs confirm in vivo findings that 2,6-dimethylaniline (DMA) is an important metabolite of lidocaine in cows, being for both PCLSs and S9 the end-product. In case of S9, also conversion of lidocaine to lidocaine-N-oxide and monoethylglycinexylidine (MEXG) was observed. MEGX is considered as intermediate for DMA formation, given that this metabolite was metabolized to DMA by both PLCSs and S9. In contrast to in vivo, no in vitro conversion of DMA to 4-OH-DMA was observed. Further work is needed to explain this lack of conversion and to further evaluate the use of slaughterhouse-derived tissue materials to predict the biotransformation of xenobiotics in farm animals.

Novel microfluidic technologies allow the manufacture of in vitro organ-on-a-chip systems that hold great promise to adequately recapitulate the biophysical and functional complexity of organs found in vivo. In this study, a gut-on-a-chip model was developed aiming to study the potential cellular association and transport of food contaminants. Intestinal epithelial cells (Caco-2) were cultured on a porous polyester membrane that was tightly clamped between two glass slides to form two separate flow chambers. Glass syringes, polytetrafluoroethylene tubing and glass microfluidic chips were selected to minimize surface adsorption of the studied compounds (i.e. highly lipophilic dioxins), during the transport studies. Confocal microscopy studies revealed that, upon culturing under constant flow for 7 days, Caco-2 cells formed complete and polarized monolayers as observed after culturing for 21 days under static conditions in Transwells. We exposed Caco-2 monolayers in the chip and Transwell to a mixture of 17 dioxin congeners (7 polychlorinated dibenzo-p-dioxins and 10 polychlorinated dibenzofurans) for 24 h. Gas chromatography-high resolution mass spectrometry was used to assess the cellular association and transport of individual dioxin congeners across the Caco-2 cell monolayers. After 24 h, the amount of transported dioxin mixture was similar in both the dynamic gut-on-a-chip model and the static Transwell model. The transport of individual congeners corresponded with their number of chlorine atoms and substitution patterns as revealed by quantitative structure-property relationship modelling. These results show that the gut-on-a-chip model can be used, as well as the traditional static Transwell system, to study the cellular association and transport of lipophilic compounds like dioxins.