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Complete proteomic datasets are available in the PRIDE partner repository (Perez-Riverol et al., 2019) under identification numbers: PXD030822 and 10.6019/PXD030822 for HepaRG cells, PXD031045 and 10.6019/PXD031045 for HepaRG culture media, PXD030887 and 10.6019/PXD030887 for PHHs, and PXD030892 and 10.6019/PXD030892 for PRHs. Metabolomic data are available in the MetaboLights repository (Haug et al., 2020) under the identification number: www.ebi.ac.uk/metabolights/MTBLS4063.; International audience; The biological effects of the pesticide and mitochondrial complex I inhibitor tebufenpyrad (TEBU) on liver cells were investigated by combining proteomics and metabolomics. Both cell culture media and cellular lysates were analyzed in dose-response and kinetic experiments on the HepaRG cell line. Responses were compared with those obtained on primary human and rat hepatocytes. A multitude of phase I and II metabolites (>80) mainly common to HepaRG cells and primary hepatocytes and an increase in metabolization enzymes were observed. Synthesis of mitochondrion and oxidative phosphorylation complex constituents, fatty acid oxidation, and cellular uptake of lipids were induced to compensate for complex I inhibition and the decrease in ATP intracellular contents caused by TEBU. Secretion of the 20 S circulating proteasome and overall inhibition of acute inflammation followed by IL-6 secretion in later stages were observed in HepaRG cells. These effects were associated with a decrease in STAT1 and STAT3 transcription factor abundances, but with different kinetics. Based on identified TEBU targets, docking experiments, and nuclear receptor reporter assays, we concluded that liver cell response to TEBU is mediated by its interaction with the PPARγ transcription factor.

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

International audience; Lipophilic phycotoxins are secondary metabolites produced by phytoplankton. They can accumulate in edible filtering-shellfish and cause human intoxications, particularly gastrointestinal symptoms. Up to now, the in vitro intestinal effects of these toxins have been mainly investigated on simple monolayers of intestinal cells such as the enterocyte-like Caco-2 cell line. Recently, the combination of Caco-2 cells with mucus secreting HT29-MTX cell line has been also used to mimic the complexity of the human intestinal epithelium. Besides, enteric glial cells (EGC) from the enteric nervous system identified in the gut mucosa have been largely shown to be involved in gut functions. Therefore, using a novel model integrating Caco-2 and HT29-MTX cells co-cultured on inserts with EGC seeded in the basolateral compartment, we examined the toxicological effects of two phycotoxins, pectenotoxin-2 (PTX2) and okadaic acid (OA). Cell viability, morphology, barrier integrity, inflammation, barrier crossing, and the response of some specific glial markers were evaluated using a broad set of methodologies. The toxicity of PTX2 was depicted by a slight decrease of viability and integrity as well as a slight increase of inflammation of the Caco-2/HT29-MTX co-cultures. PTX2 induced some modifications of EGC morphology. OA induced IL-8 release and decreased viability and integrity of Caco-2/HT29-MTX cell monolayers. EGC viability was slightly affected by OA. The presence of EGC reinforced barrier integrity and reduced the inflammatory response of the epithelial barrier following OA exposure. The release of GDNF and BDNF gliomediators by EGC could be implicated in the protection observed.

International audience; Portimine, a recently identified cyclic imine produced by the dinoflagellate Vulcanodinium rugosum, has been described as a potent apoptotic agent in contrast to most of the cyclic imines that are well-known to be neurological toxins. As apoptosis can be a consequence of a high level of DNA lesions, we investigated the responses of portimine on several endpoints aimed at detecting DNA damage in the hepatic cell line HepaRG. Portimine induced phosphorylation of H2AX, which could possibly be consistent with the previously published induction of apoptosis with this toxin. In addition, detection of apoptosis through the activation of caspase-3, the induction of strand breaks detected by the comet assay as well as chromosome and genome mutations using the micronucleus assay were addressed. Surprisingly, portimine treatment resulted in increases in only γH2AX in differentiated HepaRG cells whereas no effects on the other endpoints were detected. These increases in γH2AX in the absence of genotoxic effects in the other tests could indicate that portimine could possibly induce a DNA replication stress and/or that the compound can be detoxified by the HepaRG cells.

Manufactured nanomaterials (NMs) are increasingly used in a wide range of industrial applications leading to a constant increase in the market size of nano-enabled products. The increased production and use of NMs are raising concerns among different stakeholder groups with regard to their effects on human and environmental health. Currently, nanosafety hazard assessment is still widely performed using in vivo (animal) models, however the development of robust and reg­ulatory relevant strategies is required to prioritize and/or reduce animal testing. An adverse outcome pathway (AOP) is a structured representation of biological events that start from a molecular initiating event (MIE) leading to an adverse outcome (AO) through a series of key events (KEs). The AOP framework offers great advancement to risk assessment and regulatory safety assessments. While AOPs for chemicals have been more frequently reported, the AOP collection for NMs is limited. By using existing AOPs, we aimed to generate simple and testable strategies to predict if a given NM has the potential to induce a MIE leading to an AO through a series of KEs. Firstly, we identified potential MIEs or initial KEs reported for NMs in the literature. Then, we searched the identified MIE or initial KEs as keywords in the AOP-Wiki to find associated AOPs. Finally, using two case studies, we demonstrate how in vitro strategies can be used to test the identified MIE/KEs. ispartof: ALTEX-ALTERNATIVES TO ANIMAL EXPERIMENTATION vol:38 issue:4 pages:580-594 ispartof: location:Germany status: published

International audience; To evaluate the nanoparticle (NP) toxicity, much efforts have been devoted for developing methods to accurately disperse NPs into aqueous suspensions prior to in vitro toxicological studies. As NP toxicity is strongly dependent on their physicochemical properties, NP characterization is a key step for any in vitro toxicological study. This study demonstrates that the static multiple light scattering (SMLS) technique allows for the simultaneous screening of the NP size, agglomeration state, stability and dosimetry in biological media. Batch dispersions of TiO2 P25 NPs in water with various bovine serum albumin (BSA) mass fractions (from 0% to 0.5%) and dilutions of these dispersions into cell culture media were characterized with SMLS. In the batch dispersions, TiO2 NPs are stable and well dispersed for BSA mass fraction lower than 0.2% while agglomeration and rapid settling is observed for higher BSA mass fractions. Paradoxically, when diluted in cell culture media, TiO2 NPs are well dispersed and stable for BSA mass fractions higher than 0.2%. The TiO2 NP dosimetry of these dilutions was evaluated experimentally with SMLS and confronted with numerical approaches. The TiO2 NP bottom concentration evolves far more slowly in the case of the higher BSA mass fraction. Such measurements give valuable insights on the NP fate and transport in biological media to obtain in fine reliable size and dose-cytotoxicity responses.

International audience; Genotoxicity is one of the key endpoints investigated as early as possible before marketing a product. Several assays can be performed to evaluate the genotoxicity and mutagenicity of compounds, covering the detection of DNA lesions and of DNA repair activities as well as the quantification of the three types of mutations at the gene, chromosome and genome levels. Most of these tests have an established or draft guideline from the Organization for Economic Co-operation and Development (OECD). Nevertheless, if these assays have been developed for chemicals, they are not fully suitable for testing the genotoxicity/mutagenicity of nanomaterials, and the adaptation of the experimental conditions for such compounds is still debated. This Special Issue “From Basic Research to New Tools and Challenges for the Genotoxicity Testing of Nanomaterials” aimed to provide new results on a large range of nanomaterials using a broad panel of genotoxicity assays and to solve some of the numerous issues faced on this topic.

Nanotechnologies have reached maturity and market penetration that require nano‐specific changes in legislation and harmonization among legislation domains, such as the amendments to REACH for nanomaterials (NMs) which came into force in 2020. Thus, an assessment of the components and regulatory boundaries of NMs risk governance is timely, alongside related methods and tools, as part of the global efforts to optimise nanosafety and integrate it into product design processes, via Safe(r)‐by‐Design (SbD) concepts. This paper provides an overview of the state‐of‐the‐art regarding risk governance of NMs and lays out the theoretical basis for the development and implementation of an effective, trustworthy and transparent risk governance framework for NMs. The proposed framework enables continuous integration of the evolving state of the science, leverages best practice from contiguous disciplines and facilitates responsive re‐thinking of nanosafety governance to meet future needs. To achieve and operationalise such framework, a science‐based Risk Governance Council (RGC) for NMs is being developed. The framework will provide a toolkit for independent NMs' risk governance and integrates needs and views of stakeholders. An extension of this framework to relevant advanced materials and emerging technologies is also envisaged, in view of future foundations of risk research in Europe and globally., This study received funding from the European Union's Horizon 2020 research and innovation program under grant agreement No 814425 (RiskGONE) and No 814572 (NanoSolveIT).

International audience; Pinnatoxins (PnTXs) are a group of emerging marine biotoxins produced by the benthic dinoflagellate Vulcanodinium rugosum, currently not regulated in Europe or in any other country in the world. In France, PnTXs were detected for the first time in 2011, in mussels from the Ingril lagoon (South of France, Mediterranean coast). Since then, analyses carried out in mussels from this lagoon have shown high concentrations of PnTXs for several months each year. PnTXs have also been detected, to a lesser extent, in mussels from other Mediterranean lagoons and on the Atlantic and Corsican coasts. In the French data, the main analog is PnTX G (low levels of PnTX A are also present in some samples). No cases of PnTXs poisoning in humans have been reported so far in France or anywhere else in the world. In mice, PnTXs induce acute neurotoxic effects, within a few minutes after oral administration. Clinical signs of toxicity include decreased mobility, paralysis of the hind legs, tremors, jumps and breathing difficulties leading to death by respiratory arrest at high doses. The French agency for food safety (ANSES) recently conducted a review of the state of knowledge related to PnTXs and V. rugosum. Based on (i) the clinical signs of toxicity in mice, (ii) the mode of action of PnTXs as nicotinic acetylcholine receptor competitive antagonists and (iii) knowledge on drugs and natural toxins with PnTX-related pharmacology, potential human symptoms have been extrapolated and proposed. In this work, a provisional acute benchmark value for PnTX G of 0.13 μg/kg bw per day has been derived from an oral acute toxicity study in mice. Based on this value and a large shellfish meat portion size of 400g, a concentration lower than 23 μg PnTX G/kg shellfish meat is not expected to result in adverse effects in humans. ANSES recommends taking into account PnTXs in the French official monitoring program for shellfish production and identified data gaps to refine health risk assessment.

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

Due to several gaps remaining in the toxicological evaluation of nanomaterials (NMs), consumers and public health agencies have shown increasing concern for human health protection. In addition to aluminum (Al) microparticles, Al-containing nanomaterials (Al NMs) have been applied by food industry as additives and contact materials. Due to the limited amount of literature on the toxicity of Al NMs, this study aimed to evaluate the in vivo genotoxic potential of Al0 and Al2O3 NMs after acute oral exposure. Male Sprague-Dawley rats were administered three successive gavages at 6, 12.5 and 25 mg/kg bw. A comparison with AlCl3 was done in order to assess the potential effect of dissolution into Al ions. Both DNA strand breaks and oxidative DNA damage were investigated in six organs/tissues (duodenum, liver, kidney, spleen, blood and bone marrow) with the alkaline and the Fpg-modified comet assays. Concomitantly, chromosomal damage was investigated in bone marrow and colon with the micronucleus assay. The comet assay only showed DNA damage with Al2O3 NMs in bone marrow (BM), while AlCl3 induced slight but non-significant oxidative DNA damage in blood. No increase of chromosomal mutations was observed after treatment with the two Al MNs either in the BM or in the colons of rats.

International audience; The hepatotoxin cylindrospermopsin (CYN) has been involved in cases of poisoning in humans following ingestion. As its liver toxicity process is complex, we studied the transcriptomic profile of HepaRG cells exposed to CYN. The affected pathways were confirmed through the expression of key genes and the investigation of toxicity markers. In addition, CYP450 activities and cell redox homeostasis were investigated following acute and repeated exposure. CYN induced the down-regulation of genes involved in xenobiotic metabolism and cell cycle progression. There was cell cycle disturbance characterised by an accumulation of G1/S and G2/M cells and an increase in phospho-H3-positive cells. This was linked to the induction of DNA damage demonstrated by an increase in γH2AX-positive cells as well as an accumulation of sub-G1 cells indicating apoptosis but not involving caspase-3. While glutathione (GSH) content sharply decreased following acute exposure to CYN, it increased following repeated exposure, reflecting an adaptive response of cell redox homeostasis. However, our data also suggested that CYN induced the down-regulation of phase I and II metabolism gene products, and CYP450 activities were affected following both acute and repeated exposure to CYN. Our study indicated that repeated exposure of liver cells to low concentrations of CYN may affect their detoxification capacities.

International audience; 13-Desmethylspirolide C (13-SPX-C) is a phycotoxin produced by dinoflagellates which can accumulate in shellfish. 13-SPX-C induces neurotoxic effects in rodents through blockade of nicotinic acetylcholine receptors. As no human intoxication has been to date attributed to the consumption of 13-SPX-C-contaminated seafood, this toxin is not regulated according to the Codex Alimentarius. Nevertheless, shellfish consumers can be exposed to 13-SPX-C via shellfish consumption. In order to follow the fate of the toxin after ingestion and to verify whether metabolic detoxification could explain the lack of human intoxications, we assessed the metabolism of 13-SPX-C using several in vitro liver systems. First, both phase I and II reactions occurring with rat and human liver S9 fractions were screened. Our results indicated that 13-SPX-C was almost completely metabolized with both rat and human liver S9. Using a receptor binding assay towards nicotinic acetylcholine receptors we demonstrated that the resulting metabolites showed less affinity towards nicotinic acetylcholine receptors than 13-SPX-C. Finally, we showed that 13-SPX-C induced a pronounced increase of gene expression of the drug-metabolizing enzyme cytochrome P450 (CYP) CYP1A2. The role of this CYP in 13-SPX-C metabolism was clarified using an innovative in vitro tool, CYP1A2-Silensomes™. In summary, this study highlights that liver first-pass metabolism can contribute to the detoxification of 13-SPX-C.

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International audience; The application of appropriate analytical techniques is essential for nanomaterial (NM) characterization. In this study, we compared different analytical techniques for NM analysis. Regarding possible adverse health effects, ionic and particulate NM effects have to be taken into account. As NMs behave quite differently in physiological media, special attention was paid to techniques which are able to determine the biosolubility and complexation behavior of NMs. Representative NMs of similar size were selected: aluminum (Al 0) and aluminum oxide (Al 2 O 3), to compare the behavior of metal and metal oxides. In addition, titanium dioxide (TiO 2) was investigated. Characterization techniques such as dynamic light scattering (DLS) and nanoparticle tracking analysis (NTA) were evaluated with respect to their suitability for fast characterization of nanoparticle dispersions regarding a particle's hydrodynamic diameter and size distribution. By application of inductively coupled plasma mass spectrometry in the single particle mode (SP-ICP-MS), individual nanoparticles were quantified and characterized regarding their size. SP-ICP-MS measurements were correlated with the information gained using other characterization techniques, i.e. transmission electron microscopy (TEM) and small angle X-ray scattering (SAXS). The particle surface as an important descriptor of NMs was analyzed by X-ray diffraction (XRD). NM impurities and their co-localization with biomolecules were determined by ion beam microscopy (IBM) and confocal Raman microscopy (CRM). We conclude advantages and disadvantages of the different techniques applied and suggest options for their complementation. Thus, this paper may serve as a practical guide to particle characterization techniques.

International audience; Volume 280, Supplement 1, Pages S1-S346 (20 October 2017)Abstracts of the 53rd Congress of the European Societies of Toxicology (EUROTOX)Bratislava, Slovakia, 10th–13th September, 2017Edited by Mumtaz Iscan and Helena KandarovaP-03-02-16 Aluminum on different forms is found in several goods: food addi- tives, medication, beverage, water treatment as well as cooking utensils. EFSA has established a tolerable weekly intake (TWI) of 1 mg/kg bw for human oral exposure (EFSA, 2008). Since the 2000s, due to the increasing use of nanoparticles (NP), aluminum NPs are expected to be more largely involved in human exposure although their effects on human health has not been fully characterized. Nevertheless, aluminum was depicted to cross epithelial barriers and to include neurotoxicity and embryotoxic- ity. As aluminum NPs may not behave as Al ions, their toxic effects must be investigated. In this study, we investigated the genotoxicity of Al, Al 2 O 3 NPs and the ionic form AlCl 3 on intestine, the contact organ fol- lowing oral exposure, and on liver, the main target organ for Al accumulation using both the alkaline comet assay and the Fpg- modified comet assay for highlighting potential oxidative damage. We observed DNA damage on the human intestinal Caco2 and hepatic HepaRG cells (exposure from 0.6 to 256 g/cm 2 ). How- ever, no genotoxicity was detected in duodenum and liver of male Sprague Dawley rats after 3 gavages as well as after 28 days oral treatment with 6, 12 and 25 mg/kg bw/day. This project was funded by Agence Nationale de la Recherche (ANR-13-IS10-0005-01).

With the growing numbers of nanomaterials (NMs), there is a great demand for rapid and reliable ways of testing NM safety—preferably using in vitro approaches, to avoid the ethical dilemmas associated with animal research. Data are needed for developing intelligent testing strategies for risk assessment of NMs, based on grouping and read‐across approaches. The adoption of high throughput screening (HTS) and high content analysis (HCA) for NM toxicity testing allows the testing of numerous materials at different concentrations and on different types of cells, reduces the effect of inter‐experimental variation, and makes substantial savings in time and cost. HTS/HCA approaches facilitate the classification of key biological indicators of NM‐cell interactions. Validation of in vitro HTS tests is required, taking account of relevance to in vivo results. HTS/HCA approaches are needed to assess dose‐ and time‐dependent toxicity, allowing prediction of in vivo adverse effects. Several HTS/HCA methods are being validated and applied for NM testing in the FP7 project NANoREG, including Label‐free cellular screening of NM uptake, HCA, High throughput flow cytometry, Impedance‐based monitoring, Multiplex analysis of secreted products, and genotoxicity methods—namely High throughput comet assay, High throughput in vitro micronucleus assay, and γH2AX assay. There are several technical challenges with HTS/HCA for NM testing, as toxicity screening needs to be coupled with characterization of NMs in exposure medium prior to the test; possible interference of NMs with HTS/HCA techniques is another concern. Advantages and challenges of HTS/HCA approaches in NM safety are discussed. WIREs Nanomed Nanobiotechnol 2017, 9:e1413. doi: 10.1002/wnan.1413 For further resources related to this article, please visit the WIREs website.

International audience; PTX-2 is a marine biotoxin frequently found in shellfish that can lead to food intoxication in humans. Information regarding PTX-2 metabolism is scarce, and little is known of its effect on xenobiotic-metabolizing enzymes (XME) or its molecular pathways. The aim of this study was consequently to examine PTX-2 Phase I metabolism using rat and human liver S9 fractions, and also to assess the capability of PTX-2: (i) to modulate the gene expression of a panel of Phase I (CYP450) and II (UGT, SULT, NAT, and GST) enzymes, as well as the Phase III or 0 (ABC and SLCO) transporters in the human hepatic HepaRG cell line using qPCR; (ii) to induce specific CYP450 in HepaRG cells measured by immunolabeling detection and the measurement of the cells' activities; and (iii) to activate nuclear receptors and induce CYP promoter activities in HEK-T and HepG2 transfected cell lines using transactivation and reporter gene assay, respectively. Our results indicate that PTX-2 hydroxylation occurred with both rat and human S9 fractions. Whereas PTX-2 mostly upregulated the gene expression of CYP1A1 and 1A2, no induction of these two CYP activities was observed. Lastly, PTX-2 did not act as an agonist of CAR or PXR. Due to its effects on some key XME, more attention should be paid to possible drug–drug interactions with phycotoxins, especially as shellfish can accumulate several phycotoxins as well as other kinds of contaminants.

Aluminum has gathered toxicological attention based on relevant human exposure and its suspected hazardous potential. Nanoparticles from food supplements or food contact materials may reach the human gastrointestinal tract. Here, we monitored the physicochemical fate of aluminum-containing nanoparticles and aluminum ions when passaging an in vitro model of the human gastrointestinal tract. Small-angle X-ray scattering (SAXS), transmission electron microscopy (TEM), ion beam microscopy (IBM), secondary ion beam mass spectrometry (TOF-SIMS), and inductively coupled plasma mass spectrometry (ICP-MS) in the single-particle mode were employed to characterize two aluminum-containing nanomaterials with different particle core materials (Al0, γAl2O3) and soluble AlCl3. Particle size and shape remained unchanged in saliva, whereas strong agglomeration of both aluminum nanoparticle species was observed at low pH in gastric fluid together with an increased ion release. The levels of free aluminum ions decreased in i...

International audience; The aim of this study was to investigate (i) the cytotoxic effects of lipophilic phycotoxins, including okadaic acid (OA) and dinophysistoxin-1 and -2 (DTX-1 and DTX-2), pectenotoxin-2 (PTX-2), yessotoxin (YTX), spirolide (SPX), and azaspiracids-1, -2 and -3 (AZA-1, AZA-2 and AZA-3), in human HepaRG cells using a multiparametric high content analysis approach, (ii) the ability of nine lipophilic phycotoxins to act as PXR agonists in a HepG2-PXR cell line, (iii) their potential to induce CYP450 activity, and (iv) the role of CYP3A4 in cytotoxicity induced by lipophilic phycotoxins. Our results indicate that while OA, DTX-1 and DTX-2 activated PXR-dependent transcriptional activity in HepG2 cells, no increase of CYP450 (1A2, 3A4, 2C9, 2C19) activities were observed in HepaRG cell following a 72h treatment with these toxins. Multiparametric analysis showed that OA, DTX-1, DTX-2, and PTX-2 were highly cytotoxic in HepaRG cells; inducing cell loss, activation of caspase-3 and gamma-H2AX formation. However, no toxicity was observed for YTX, SPX, and AZAs. Moreover, we found that inhibition of CYP3A4 activity by ketoconazole enhances the toxic effects of OA, DTX-1, DTX-2, and PTX-2 in HepaRG cells. Taken together, these results suggest that CYP3A4-mediated metabolism of some lipophilic phycotoxins decreases their in vitro toxicity.

International audience; Silica (SiO2) in its nanosized form is now used in food applications although the potential risks for human health need to be evaluated in further detail. In the current study, the uptake of 15 and 55 nm colloidal SiO2 NPs in the human intestinal Caco-2 cell line was investigated by transmission electron microscopy. The ability of these NPs to induce cytotoxicity (XTT viability test), genotoxicity (γH2Ax and micronucleus assay), apoptosis (caspase 3), oxidative stress (oxidation of 2,7-dichlorodihydrofluorescein diacetate probe) and proinflammatory effects (interleukin IL-8 secretion) was evaluated. Quartz DQ12 was used as particle control. XTT and cytokinesis-block micronucleus assays revealed size- and concentration-dependent effects on cell death and chromosome damage following exposure to SiO2 nanoparticles, concomitantly with generation of reactive oxygen species (ROS), SiO2-15 nm particles being the most potent. In the same way, an increased IL-8 secretion was only observed with SiO2-15 nm at the highest tested dose (32 μg/ml). TEM images showed that both NPs were localized within the cytoplasm but did not enter the nucleus. SiO2-15 nm, and to a lower extent SiO2-55 nm, exerted toxic effects in Caco-2 cells. The observed genotoxic effects of these NPs are likely to be mediated through oxidative stress rather than a direct interaction with the DNA. Altogether, our results indicate that exposure to SiO2 NPs may induce potential adverse effects on the intestinal epithelium in vivo.

Deoxynivalenol (DON) is the major mycotoxin detected in cereal foods and a risk for human health following DON ingestion could not be excluded due to high level exposure. In this light, the hazard of DON must be carefully evaluated. Therefore, the aim of this study is to perform in vitro genotoxicity tests with DON using the Salmonella typhimurium reverse mutation assay (Ames’ test), the comet assay and the micronucleus test in accordance with the OECD test guideline 487 in two human cell lines: the lymphoblastoid TK6 and the hepatoma HepaRG cells. DON gave negative results in the Ames’ test performed, both with and without rat liver S9 on three strains TA98, TA100 and TA102. DON elicited cytotoxicity in TK6 and HepaRG cells but did not induce primary DNA damage. DON failed also to induce MN formation in TK6 cells with or without human and rat liver S9. After 24 h of treatment, DON induced micronucleus formation in TK6 cells but only at concentrations producing more than 55 ± 5% cytotoxicity. In HepaRG cells, DON highly increased the caspase-3/7 activity but no micronucleus induction was observed. Taken together, our results suggest that DON could be considered as a non in vitro genotoxin.

Brevetoxin B is a highly reactive molecule, due in part to an α,β-unsaturated aldehyde group at the terminal side chain, leading to metabolism by reduction, oxidation and conjugation. These reactions have little effect to reduce intrinsic activity at the voltage-gated sodium channel or during toxicity testing by either enzyme-linked immunosorbent assay or mouse bioassay. Here we investigate the potential for intestinal absorption of the two most abundant brevetoxins present in Gulf of Mexico oysters using human Caco-2 cell monolayers, a widely utilized in vitro test to predict oral bioavailability of drugs and their metabolites. We found that both dihydrobrevetoxin B and cysteine brevetoxin B were rapidly taken up by the Caco-2 monolayer. However, only dihydrobrevetoxin B passes through the monolayer to reach the basal compartment. Dihydrobrevetoxin B has a moderate apparent permeability coefficient of 1.6 × 10 −6 cm/s at 500 ng/mL and nearly 50% of the toxin passes from the apical to basal compartment in 24 h. The inability of the cysteine brevetoxin B to pass through an intestinal epithelial barrier suggests that this bioactive brevetoxin metabolite that persists in shellfish may not contribute to neurotoxic shellfish poisoning.

International audience; The phycotoxin, okadaic acid (OA) and dinophysistoxin 1 and 2 (DTX-1 and -2) are protein phosphatase PP2A and PP1 inhibitors involved in diarrhetic shellfish poisoning (DSP). Data on the toxicity of the OA-group toxins show some differences with respect to the in vivo acute toxicity between the toxin members. In order to investigate whether OA and congeners DTX-1 and -2 may induce different mechanisms of action during acute toxicity on the human intestine, we compared their toxicological effects in two in vitro intestinal cell models: the colorectal adenocarcinoma cell line, Caco-2, and the intestinal muco-secreting cell line, HT29-MTX. Using a high content analysis approach, we evaluated various cytotoxicity parameters, including apoptosis (caspase-3 activation), DNA damage (phosphorylation of histone H2AX), inflammation (translocation of NF-κB) and cell proliferation (Ki-67 production). Investigation of the kinetics of the cellular responses demonstrated that the three toxins induced a pro-inflammatory response followed by cell cycle disruption in both cell lines, leading to apoptosis. Our results demonstrate that the three toxins induce similar effects, as no major differences in the cytotoxic responses could be detected. However DTX-1 induced cytotoxic effects at five-fold lower concentrations than for OA and DTX-2.

International audience; Aromatic amines and structurally related heterocyclic aromatic amines (HAAs) are produced during the combustion of tobacco or during the high-temperature cooking of meat. Exposure to some of these chemicals may contribute to the etiology of several common types of human cancers. 2-Amino-9H-pyrido[2,3-b]indole (AαC) is the most abundant HAA formed in mainstream tobacco smoke: it arises in amounts that are 25-100 times greater than the levels of the arylamine, 4-aminobiphenyl (4-ABP), a human carcinogen. 2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) is a prevalent HAA formed in cooked meats. AαC and MeIQx are rodent carcinogens; however, their carcinogenic potency in humans is unknown. A preliminary assessment of the carcinogenic potential of these HAAs in humans was conducted by examining the capacity of primary human hepatocytes to form DNA adducts of AαC and MeIQx, in comparison to 4-ABP, followed by the kinetics of DNA adduct removal by cellular enzyme repair systems. The principal DNA adducts formed were N-(deoxyguanosin-8-yl) (dG-C8) adducts. Comparable levels of DNA adducts were formed with AαC and 4-ABP, whereas adduct formation was ∼5-fold lower for MeIQx. dG-C8-AαC and dG-C8-4-ABP were formed at comparable levels in a concentration-dependent manner in human hepatocytes treated with procarcinogens over a 10,000-fold concentration range (1 nM-10 μM). Pretreatment of hepatocytes with furafylline, a selective inhibitor of cytochrome P450 1A2, resulted in a strong diminution of DNA adducts signifying that P450 1A2 is a major P450 isoform involved in bioactivation of these procarcinogens. The kinetics of adduct removal varied for each hepatocyte donor. Approximately half of the DNA adducts were removed within 24 h of treatment; however, the remaining lesions persisted over 5 days. The high levels of AαC present in tobacco smoke and its propensity to form persistent DNA adducts in human hepatocytes suggest that AαC can contribute to DNA damage and the risk of hepatocellular cancer in smokers.

Due to the broad spectrum of pesticide usages, consumers are exposed to mixtures of residues, which may have combined effects on human health. The PERICLES research program aims to test the potential combined effects of pesticide mixtures, which are likely to occur through dietary exposure. The co-exposure of the French general population to 79 pesticide residues present in the diet was first assessed. A Bayesian nonparametric model was then applied to define the main mixtures to which the French general population is simultaneously and most heavily exposed. Seven mixtures made of two to six pesticides were identified from the exposure assessment. An in vitro approach was used for investigating the toxicological effects of these mixtures and their corresponding individual compounds, using a panel of cellular models, i.e. primary rat and human hepatocytes, liver, intestine, kidney, colon and brain human cell lines. A set of cell functions and corresponding end-points were monitored such as cytotoxicity, real-time cell impedance, genotoxicity, oxidative stress, apoptosis and PXR nuclear receptor transactivation. The mixtures were tested in equimolar concentrations. Among the seven mixtures, two appeared highly cytotoxic, five activated PXR and depending on the assay one or two were genotoxic. In some experiments, the mixture effect was quantitatively different from the effect expected from the addition concept. The PERICLES program shows that, for the most pesticides mixtures to which the French general population is exposed, the toxic effects observed on human cells cannot be easily predicted based on the toxic potential of each compound. Consequently, additional studies should be carried on in order to more accurately define the mixtures of chemicals to which the consumers are exposed, as well as to improve the investigation, prediction and monitoring of their potential human health effects.

Pinnatoxin G (PnTX-G) is a marine toxin belonging to the class of cyclic imines and produced by the dinoflagellate Vulcanodinium rugosum. In spite of its strong toxicity to mice, leading to the classification of pinnatoxins into the class of “fast-acting toxins”, its hazard for human health has never been demonstrated. In this study, crude extracts of V. rugosum exhibited significant cytotoxicity against Neuro2A and KB cells. IC50 values of 0.38 µg mL−1 and 0.19 µg mL−1 were estimated on Neuro2A cells after only 24 h of incubation and on KB cells after 72 h of incubation, respectively. In the case of Caco-2 cells 48 h after exposure, the crude extract of V. rugosum induced cell cycle arrest accompanied by a dramatic increase in double strand DNA breaks, although only 40% cytotoxicity was observed at the highest concentration tested (5 µg mL−1). However, PnTX-G was not a potent cytotoxic compound as no reduction of the cell viability was observed on the different cell lines. Moreover, no effects on the cell cycle or DNA damage were observed following treatment of undifferentiated Caco-2 cells with PnTX-G. The crude extract of V. rugosum was thus partially purified using liquid-liquid partitioning and SPE clean-up. In vitro assays revealed strong activity of some fractions containing no PnTX-G. The crude extract and the most potent fraction were evaluated using full scan and tandem high resolution mass spectrometry. The dereplication revealed the presence of a major compound that could be putatively annotated as nakijiquinone A, N-carboxy-methyl-smenospongine or stachybotrin A, using the MarinLit™ database. Further investigations will be necessary to confirm the identity of the compounds responsible for the cytotoxicity and genotoxicity of the extracts of V. rugosum.

Although human populations may be constantly exposed to complex pesticide mixtures through their diet, the human health risk of pesticide exposure is currently assessed on the basis of toxicity data on individual compounds. To investigate the combined toxic effects of pesticide cocktails previously identified in the French diet, we first studied the cytotoxicity induced by seven cocktails composed of two to six pesticides on human hepatic (HepG2) and colon (Caco-2) cell lines using the MTT and neutral red uptake assays. Secondly, we challenged to assess the combined effects of the two most cytotoxic cocktails by comparing the measured effects of the mixtures with the predictions based on additive effects on two concepts-independent action (IA) and concentration addition (CA). For the cocktail composed of dichlorodiphenyltrichloroethane (DDT) and dieldrin, the cytotoxicity of the equimolar cocktail proved greater than the additive effect estimated by the two concepts. Furthermore, apoptosis induction was higher in equimolar cocktail than predicted by summing the effects of DDT and dieldrin. Thus, some supra-additive toxicity was found in the DDT-dieldrin cocktail. Nevertheless, if IA and CA models could reveal combined effects of pesticide cocktails, an accurate evaluation remains challenging.

Here, we assessed a co-culture system of intestinal Caco-2 cells and lymphoblastoid TK6 cells for modelling the role of intestinal first-pass effects, i.e. absorption and metabolism, in the genotoxicity of oral drugs and food contaminants. Caco-2 cells were seeded onto semipermeable culture inserts for 21 days until differentiation, and then TK6 cells were added to the basal compartment. After apical loading with mutagenic compounds [methylmethanesulfonate (MMS), benzo[a]-pyrene (BaP) and aflatoxin B1 (AFB1)], comet and micronucleus assays were performed on both cell lines. MMS (10 µg/ml) showed positive results in the micronucleus assays in both cell lines, even though DNA damage was only detected in the Caco-2 cells with the comet assay. At concentrations of 0.5-50 µM, BaP induced dose-dependent comet and micronucleus formation at 24h in Caco-2 cells, but no DNA damage was observed in TK6 cells. Although AFB1 failed to induce comet formation, it resulted in a high level of micronuclei in both cell lines. Treatment of Caco-2 cells with the CYP3A4 inhibitor, ketoconazole, inhibited the AFB1-induced cytotoxicity and micronucleus formation in TK6 cells, suggesting that intestinal metabolism is involved in the AFB1 genotoxic response in TK6 cells. Our results suggest that the Caco-2/TK6 co-culture model is suitable for modelling the role of intestinal biotransformation and transport processes in the genotoxic potential of oral drugs and food contaminants in target blood cells.

Cylindrospermopsin (CYN), a cyanobacterial hepatotoxin mainly produced by Cylindrospermopsis raciborskii, has been involved in human intoxications and livestock deaths. The widespread occurrence of CYN in the water supplies lead us to investigate its genotoxicity to assess potential chronic effects. This study reports evaluation of CYN-induced in vivo DNA damage in mice using alkaline comet assay (ACA) and micronucleus assay (MNA) concomittantly. ACA measures DNA breakage from single and double strand breaks as well as alkali labile sites. Conversely, MNA detects chromosome damage events such as chromosomal breakage and numeric alterations. Male Swiss mice were treated with CYN concentrations of 50, 100, and 200 μg/kg by a single intraperitoneal (ip) injection or with 1, 2, and 4 mg/kg by gavage. Methyl methane sulfonate (MMS) was used as positive control at 80 mg/kg. Twenty-four hours after treatment, samples of liver, blood, bone marrow, kidney, intestine, and colon were taken to perform ACA, the bone marrow and the colon were also used for MNA. Parameters used to quantify DNA damage were % Tail DNA for ACA and both micronucleated immature erythrocytes and epithelial colon cells for MNA. DNA breaks and chromosome damage were significantly increased by MMS in all the organs evaluated. Significant DNA damage was detected within the colon by ACA after ip injection of 100 and 200 μg/kg CYN (P < 0.01). DNA damage was also detected in colon samples after 4 mg/kg oral administration of CYN and in bone marrow after 1 and 2 mg/kg of orally administered CYN. Histological examination showed foci of cell death within the liver and the kidney from mice that received the two highest doses of CYN by either route of administration. © 2010 Wiley Periodicals, Inc. Environ Toxicol, 2012.

Human poisoning due to consumption of seafood contaminated with phycotoxins is a worldwide problem, and routine monitoring programs have been implemented in various countries to protect human consumers. Following successive episodes of unexplained shellfish toxicity since 2005 in the Arcachon Bay on the French Atlantic coast, a national research program was set up to investigate these atypical toxic events. Part of this program was devoted to fit-for-purpose cell-based assays (CBA) as complementary tools to collect toxicity data on atypical positive-mouse bioassay shellfish extracts. A collaborative study involving five laboratories was conducted. The responses of human hepatic (HepG2), human intestinal (Caco2), and mouse neuronal (Neuro2a) cell lines exposed to three known lipophilic phycotoxins-okadaic acid (OA), azaspiracid-1 (AZA1), and pectenotoxin-2 (PTX2)-were investigated. A screening strategy composed of standard operating procedures and a decision tree for dose-response modeling and assay validation were designed after a round of "trial-and-error" process. For each toxin, the shape of the concentration-response curves and the IC(50) values were determined on the three cell lines. Whereas OA induced a similar response irrespective of the cell line (complete sigmoid), PTX2 was shown to be less toxic. AZA1 induced cytotoxicity only on HepG2 and Neuro2a, but not on Caco2. Intra- and inter-laboratory coefficients of variation of cell responses were large, with mean values ranging from 35 to 54 % and from 37 to 48 %, respectively. Investigating the responses of the selected cell lines to well-known toxins is the first step supporting the use of CBA among the panel of methods for characterizing atypical shellfish toxicity. Considering these successful results, the CBA strategy will be further applied to extracts of negative, spiked, and naturally contaminated shellfish tissues.

Successive unexplained shellfish toxicity events have been observed in Arcachon Bay (Atlantic coast, France) since 2005. The positive mouse bioassay (MBA) revealing atypical toxicity did not match the phytoplankton observations or the liquid chromatography-tandem mass spectrometry (LC-MS/MS) investigations used to detect some known lipophilic toxins in shellfish. The use of the three cell lines (Caco2, HepG2, and Neuro2a) allows detection of azaspiracid-1 (AZA1), okadaic acid (OA), or pectenotoxin-2 (PTX2). In this study, we proposed the cell-based assays (CBA) as complementary tools for collecting toxicity data about atypical positive MBA shellfish extracts and tracking their chromatographic fractionation in order to identify toxic compound(s). The present study was intended to investigate the responses of these cell lines to shellfish extracts, which were either control or spiked with AZA1, OA, or PTX2 used as positive controls. Digestive glands of control shellfish were extracted using the procedure of the standard MBA for lipophilic toxins and then tested for their cytotoxic effects in CBA. The same screening strategy previously used with pure lipophilic toxins was conducted for determining the intra- and inter-laboratory variabilities of the responses. Cytotoxicity was induced by control shellfish extracts whatever the cell line used and regardless of the geographical origin of the extracts. Even though the control shellfish extracts demonstrated some toxic effects on the selected cell lines, the extracts spiked with the selected lipophilic toxins were significantly more toxic than the control ones. This study is a crucial step for supporting that cell-based assays can contribute to the detection of the toxic compound(s) responsible for the atypical toxicity observed in Arcachon Bay, and which could also occur at other coastal areas.