Novel Insights on the Toxicity of Phycotoxins on the Gut through the Targeting of Enteric Glial Cells

International audienceIn vitro and in vivo studies have shown that phycotoxins can impact intestinal epithelial cells and can cross the intestinal barrier to some extent. Therefore, phycotoxins can reach cells underlying the epithelium, such as enteric glial cells (EGCs), which are involved in gut homeostasis, motility, and barrier integrity. This study compared the toxicological effects of pectenotoxin-2 (PTX2), yessotoxin (YTX), okadaic acid (OA), azaspiracid-1 (AZA1), 13-desmethyl-spirolide C (SPX), and palytoxin (PlTX) on the rat EGC cell line CRL2690. Cell viability, morphology, oxidative stress, inflammation, cell cycle, and specific glial markers were evaluated using RT-qPCR and high content analysis (HCA) approaches. PTX2, YTX, OA, AZA1, and PlTX induced neurite alterations, oxidative stress, cell cycle disturbance, and increase of specific EGC markers. An inflammatory response for YTX, OA, and AZA1 was suggested by the nuclear translocation of NF-κB. Caspase-3-dependent apoptosis and induction of DNA double strand breaks (γH2AX) were also observed with PTX2, YTX, OA, and AZA1. These findings suggest that PTX2, YTX, OA, AZA1, and PlTX may affect intestinal barrier integrity through alterations of the human enteric glial system. Our results provide novel insight into the toxicological effects of phycotoxins on the gut

Three-dimensional HepaRG spheroids as a liver model to study human genotoxicity in vitro with the single cell gel electrophoresis assay

International audienceMany efforts have been made in the last 30 years to develop more relevant in vitro models to study genotoxic responses of drugs and environmental contaminants. While 2D HepaRG cells are one of the most promising models for liver toxicology, a switch to 3D cultures that integrate both in vivo architecture and cell-cell interactions has occurred to achieve even more predictive models. preliminary studies have indicated that 3D HepaRG cells are suitable for liver toxicity screening. Our study aimed to evaluate the response of HepaRG spheroids exposed to various genotoxic compounds using the single cell gel electrophoresis assay. HepaRG spheroids were used at 10 days after seeding and exposed for 24 and 48 hours to certain selected chemical compounds (methylmethansulfonate (MMS), etoposide, benzo[a]pyrene (B[a]P), cyclophosphamide (CPA), 7,12-dimethylbenz[a]anthracene (DMBA), 2-acetylaminofluorene (2-AAF), 4-nitroquinoline (4-NQO), 2-amino-1-methyl-6-phenylimidazo[4,5-b] pyridine (PhIP), 2-amino-3-methylimidazo[4,5-f]quinolone (IQ), acrylamide, and 2-4-diaminotoluene (2,4-DAT)). After treatment, the comet assay was performed on single cell suspensions and cytotoxicity was determined by the ATP assay. Comet formation was observed for all compounds except IQ, etoposide and 2,4-DAT. Treatment of spheroids with rifampicin increased CYP3A4 activity, demonstrating the metabolic capacity of HepaRG spheroids. These data on genotoxicity in 3D HepaRG spheroids are promising, but further experiments are required to prove that this model can improve the predictivity of in vitro models to detect human carcinogens

Simultaneous Detection of 14 Microcystin Congeners from Tissue Samples Using UPLC- ESI-MS/MS and Two Different Deuterated Synthetic Microcystins as Internal Standards

International audienceCyanobacterial microcystins (MCs), potent serine/threonine-phosphatase inhibitors, pose an increasing threat to humans. Current detection methods are optimised for water matrices with only a few MC congeners simultaneously detected. However, as MC congeners are known to differ in their toxicity, methods are needed that simultaneously quantify the congeners present, thus allowing for summary hazard and risk assessment. Moreover, detection of MCs should be expanded to complex matrices, e.g., blood and tissue samples, to verify in situ MC concentrations, thus providing for improved exposure assessment and hazard interpretation. To achieve this, we applied two synthetic deuterated MC standards and optimised the tissue extraction protocol for the simultaneous detection of 14 MC congeners in a single ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) run. This procedure was validated using plasma and liver homogenates of mice (male and female) spiked with deuterated MC standards. For proof of concept, tissue and plasma samples from mice i.p. injected with MC-LR and MC-LF were analysed. While MC-LF was detected in all tissue samples of both sexes, detection of MC-LR was restricted to liver samples of male mice, suggesting different toxicokinetics in males, e.g., transport, conjugation or protein binding. Thus, deconjugation/-proteinisation steps should be employed to improve detection of bound MC. Key Contribution: The use of deuterated microcystin standards and an improved extraction procedure using UPLC-MS/MS analytics, provides for the simultaneous detection of fourteen microcystin congeners. Thus, it allows more accurate quantitation of total microcystin load of a given sample in complex matrices like blood or tissue, and therefore better hazard interpretation

Hazard assessment of nanomaterials using in vitro toxicity assays:Guidance on potential assay interferences and mitigating actions to avoid biased results

The movement towards an animal-free testing approach for risk assessment represents a key paradigm shift in toxicology. Risk assessment of engineered and anthropogenic nanoscale materials (NM) is dependent on reliable hazard characterization, which requires validated test methods and models, and increasingly on mechanistic insights into the mode of action. The properties that make NMs so advantageous for a wide range of commercial and industrial applications also pose a challenge when it comes to safety testing under in vitro and in chemico experimental settings. Their large reactive surface area makes NMs prone to interactions with assay reagents, readout signals, or intermediate steps of many test assays, leading to the potential for biased results and data inconsistencies, collectively referred to as interferences. Therefore, methods and protocols developed and validated for conventional chemicals often require adaptation and checking for reliability in NMs' toxicity assessment. This review presents the collected scientific knowledge on NMs-induced interferences for the most common in vitro toxicity assays and methods related to cytotoxicity, oxidative stress and inflammatory response evaluation. Our analysis of existing scientific literature showed that the challenge of NMs-induced interference was not explicitly addressed in more than 90% of the papers published up to 2014 reporting the safety and toxicity of NMs. In later years, increasing number of studies tackled the interference challenge in toxicity testing of NMs, which initiated exhaustive work on standardization and validation of existing regulatory-relevant in vitro test protocols and guidelines. Due to the specificity of the different NMs and the range of ways they can potentially interfere with in vitro assays, interference and fit-for purpose controls should be included for each NM type and method applied, unless label-free assays are selected. Here, we provide a decision tree to guide researchers on how to design experiments to avoid interferences during in vitro testing by taking appropriate mitigation actions and how to include proper interference controls in their experimental design where complete avoidance is not possible. The application of this decision tree will improve the reliability, comparability and reusability of in vitro toxicity data on engineered NMs or ENMs, increasing the relevance of in silico hazard data for use in risk assessment and in science-based risk governance of NMs. The approach is applicable more broadly also, to advanced materials and to hazard assessment of anthropogenic nanoscale materials such as microplastic and tyre-wear particles

Hazard assessment of nanomaterials using in vitro toxicity assays:Guidance on potential assay interferences and mitigating actions to avoid biased results

The movement towards an animal-free testing approach for risk assessment represents a key paradigm shift in toxicology. Risk assessment of engineered and anthropogenic nanoscale materials (NM) is dependent on reliable hazard characterization, which requires validated test methods and models, and increasingly on mechanistic insights into the mode of action. The properties that make NMs so advantageous for a wide range of commercial and industrial applications also pose a challenge when it comes to safety testing under in vitro and in chemico experimental settings. Their large reactive surface area makes NMs prone to interactions with assay reagents, readout signals, or intermediate steps of many test assays, leading to the potential for biased results and data inconsistencies, collectively referred to as interferences. Therefore, methods and protocols developed and validated for conventional chemicals often require adaptation and checking for reliability in NMs' toxicity assessment. This review presents the collected scientific knowledge on NMs-induced interferences for the most common in vitro toxicity assays and methods related to cytotoxicity, oxidative stress and inflammatory response evaluation. Our analysis of existing scientific literature showed that the challenge of NMs-induced interference was not explicitly addressed in more than 90% of the papers published up to 2014 reporting the safety and toxicity of NMs. In later years, increasing number of studies tackled the interference challenge in toxicity testing of NMs, which initiated exhaustive work on standardization and validation of existing regulatory-relevant in vitro test protocols and guidelines. Due to the specificity of the different NMs and the range of ways they can potentially interfere with in vitro assays, interference and fit-for purpose controls should be included for each NM type and method applied, unless label-free assays are selected. Here, we provide a decision tree to guide researchers on how to design experiments to avoid interferences during in vitro testing by taking appropriate mitigation actions and how to include proper interference controls in their experimental design where complete avoidance is not possible. The application of this decision tree will improve the reliability, comparability and reusability of in vitro toxicity data on engineered NMs or ENMs, increasing the relevance of in silico hazard data for use in risk assessment and in science-based risk governance of NMs. The approach is applicable more broadly also, to advanced materials and to hazard assessment of anthropogenic nanoscale materials such as microplastic and tyre-wear particles

Simultaneous Quantification and Visualization of Titanium Dioxide Nanomaterial Uptake at the Single Cell Level in an In Vitro Model of the Human Small Intestine

International audienceUseful properties render titanium dioxide nanomaterials (NMs) to be one of the most commonly used NMs worldwide. TiO2 powder is used as food additives (E171), which may contain up to 36% nanoparticles. Consequently, humans could be exposed to comparatively high amounts of NMs that may induce adverse effects of chronic exposure conditions. Visualization and quantification of cellular NM uptake as well as their interactions with biomolecules within cells are key issues regarding risk assessment. Advanced quantitative imaging tools for NM detection within biological environments are therefore required. A combination of the label-free spatially resolved dosimetric tools, microresolved particle induced X-ray emission and Rutherford backscattering, together with high resolution imaging techniques, such as time-of-flight secondary ion mass spectrometry and transmission electron microscopy, are applied to visualize the cellular translocation pattern of TiO2 NMs and to quantify the NM-load, cellular major, and trace elements in differentiated Caco-2 cells as a function of their surface properties at the single cell level. Internalized NMs are not only able to impair the cellular homeostasis by themselves, but also to induce an intracellular redistribution of metabolically relevant elements such as phosphorus, sulfur, iron, and copper

Maitotoxin-4, a Novel MTX Analog Produced by Gambierdiscus excentricus

Maitotoxins (MTXs) are among the most potent toxins known. These toxins are produced by epi-benthic dinoflagellates of the genera Gambierdiscus and Fukuyoa and may play a role in causing the symptoms associated with Ciguatera Fish Poisoning. A recent survey revealed that, of the species tested, the newly described species from the Canary Islands, G. excentricus, is one of the most maitotoxic. The goal of the present study was to characterize MTX-related compounds produced by this species. Initially, lysates of cells from two Canary Island G. excentricus strains VGO791 and VGO792 were partially purified by (i) liquid-liquid partitioning between dichloromethane and aqueous methanol followed by (ii) size-exclusion chromatography. Fractions from chromatographic separation were screened for MTX toxicity using both the neuroblastoma neuro-2a (N2a) cytotoxicity and Ca2+ flux functional assays. Fractions containing MTX activity were analyzed using liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS) to pinpoint potential MTX analogs. Subsequent non-targeted HRMS analysis permitted the identification of a novel MTX analog, maitotoxin-4 (MTX4, accurate mono-isotopic mass of 3292.4860 Da, as free acid form) in the most toxic fractions. HRMS/MS spectra of MTX4 as well as of MTX are presented. In addition, crude methanolic extracts of five other strains of G. excentricus and 37 other strains representing one Fukuyoa species and ten species, one ribotype and one undetermined strain/species of Gambierdiscus were screened for the presence of MTXs using low resolution tandem mass spectrometry (LRMS/MS). This targeted analysis indicated the original maitotoxin (MTX) was only present in one strain (G. australes S080911_1). Putative maitotoxin-2 (p-MTX2) and maitotoxin-3 (p-MTX3) were identified in several other species, but confirmation was not possible because of the lack of reference material. Maitotoxin-4 was detected in all seven strains of G. excentricus examined, independently of their origin (Brazil, Canary Islands and Caribbean), and not detected in any other species. MTX4 may therefore serve as a biomarker for the highly toxic G. excentricus in the Atlantic area

Hazard characterization of Alternaria toxins to identify data gaps and improve risk assessment for human health

Fungi of the genus Alternaria are ubiquitous plant pathogens and saprophytes which are able to grow under varying temperature and moisture conditions as well as on a large range of substrates. A spectrum of structurally diverse secondary metabolites with toxic potential has been identified, but occurrence and relative proportion of the different metabolites in complex mixtures depend on strain, substrate, and growth conditions. This review compiles the available knowledge on hazard identification and characterization of Alternaria toxins. Alternariol (AOH), its monomethylether AME and the perylene quinones altertoxin I (ATX-I), ATX-II, ATX-III, alterperylenol (ALP), and stemphyltoxin III (STTX-III) showed in vitro genotoxic and mutagenic properties. Of all identified Alternaria toxins, the epoxide-bearing analogs ATX-II, ATX-III, and STTX-III show the highest cytotoxic, genotoxic, and mutagenic potential in vitro. Under hormone-sensitive conditions, AOH and AME act as moderate xenoestrogens, but in silico modeling predicts further Alternaria toxins as potential estrogenic factors. Recent studies indicate also an immunosuppressive role of AOH and ATX-II; however, no data are available for the majority of Alternaria toxins. Overall, hazard characterization of Alternaria toxins focused, so far, primarily on the commercially available dibenzo-α-pyrones AOH and AME and tenuazonic acid (TeA). Limited data sets are available for altersetin (ALS), altenuene (ALT), and tentoxin (TEN). The occurrence and toxicological relevance of perylene quinone-based Alternaria toxins still remain to be fully elucidated. We identified data gaps on hazard identification and characterization crucial to improve risk assessment of Alternaria mycotoxins for consumers and occupationally exposed workers.The European Partnership for the Assessment of Risks from Chemicals has received funding from the European Union’s Horizon Europe research and innovation program under Grant Agreement No 101057014 and has received co-funding of the authors’ institutions. Views and opinions expressed are, however, those of the author(s) only and do not necessarily reflect those of the European Union or the Health and Digital Executive Agency. Neither the European Union nor the granting authority can be held responsible for them.info:eu-repo/semantics/publishedVersio

From Basic Research to New Tools and Challenges for the Genotoxicity Testing of Nanomaterials

Genotoxicity is one of the key endpoints investigated as early as possible before marketing a product [...