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Beauvericin (BEA) and Enniatins (ENN) are mycotoxins produced by Fusarium fungi detected in food and feed; there are insufficient data to establish their reference values. To evaluate BEA and ENN oral toxicity, an integrated approach was applied. Among ENN, Enniatin B (ENNB) was selected as test substance. The approach is composed by: i) in vitro and acute in vivo genotoxicity tests; ii) a repeated-dose oral toxicity study focused on genotoxic, immune, endocrine, nervous endpoints and the reproductive/developmental toxicity screening. For BEA, all the genotoxicity endpoints yielded negative results excluding Comet assay in duodenum and kidney after repeated doses. BEA immunotoxicity was observed in female mice, concentrated in number and functional activity of effector T cells in the spleen. Based on the repeated-dose BEA study, the No Observed Adverse Effect Level (NOAEL) for female mice is 1 mg/kg b.w. per day (increased thyroid pycnotic nuclei and endometrial hyperplasia). In males, the NOAEL is 0.1 mg/kg b.w. per day (reduced colloid and altered T4 serum levels). Maternal NOAEL is 0.1 mg/kg b.w. per day (increased thymus weight), developmental NOAEL is 10 mg/kg b.w. per day. For ENNB, the results support a genotoxic effect in bone marrow and liver cells after acute treatment, but not after repeated exposure. Immunotoxic ENNB effects were observed in both genders, suggestive of a suppressive/inhibiting activity particularly evident in males. Based on the repeated-dose ENNB study, the NOAEL for females is 0.18 mg/kg b.w. per day (histomorphometrical effects on thymus, uterus and spleen). In male mice, the NOAEL is 1.8 mg/kg b.w. per day (enterocyte vacuolization in duodenum and increased Reactive Oxygen Species and reduced Glutathione brain levels). The maternal NOAEL is 1.8 mg/kg b.w. per day (decreased white pulp area and increased red/white pulp area ratio in spleen), developmental NOAEL is 18 mg/kg b.w. per day.

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.

A review of in vitro genotoxicity studies on titanium dioxide nanoparticles (TiO

IF 2.047; International audience; Food contact paperboards may be a potential source of food contamination as they can release chemicals (intentionally added or not), especially recycled paperboards. In this study, we assessed the in vitro genotoxicity of food contact paperboard samples from a manufacturer, collected at the beginning and at the end of a recycling production chain. Samples were extracted in water to mimic a wet food contact. Different genotoxic endpoints were evaluated in two human hepatic cell lines (HepG2 and HepaRG) using bioassays: γH2AX and p53 activation, primary DNA damage with the comet assay and micronucleus formation. We found that the samples from the beginning and the end of the production chain induced, with the same potency, γH2AX and p53-ser15 activation and DNA damage with the comet assay. The micronucleus assay was negative with the paperboard extract from the beginning of the chain, whereas positive data were observed for the end paperboard extract. Our results indicate that samples from recycled food contact paperboard can induce in vitro genotoxic effects in our experimental conditions.

International audience; Development and market introduction of new nanomaterials trigger the need for an adequate risk assessment of such products alongside suitable risk communication measures. Current application of classical and new nanomaterials is analyzed in context of regulatory requirements and standardization for chemicals, food and consumer products. The challenges of nanomaterial characterization as the main bottleneck of risk assessment and regulation are presented. In some areas, e.g., quantification of nanomaterials within complex matrices, the establishment and adaptation of analytical techniques such as laser ablation inductively coupled plasma mass spectrometry and others are potentially suited to meet the requirements. As an example, we here provide an approach for the reliable characterization of human exposure to nanomaterials resulting from food packaging. Furthermore, results of nanomaterial toxicity and ecotoxicity testing are discussed, with concluding key criteria such as solubility and fiber rigidity as important parameters to be considered in material development and regulation. Although an analysis of the public opinion has revealed a distinguished rating depending on the particular field of application, a rather positive perception of nanotechnology could be ascertained for the German public in general. An improvement of material characterization in both toxicological testing as well as end-product control was concluded as being the main obstacle to ensure not only safe use of materials, but also wide acceptance of this and any novel technology in the general public

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...

Toxic algal blooms are a raising concern all over the world due to their expansion during the last 10 years. Two main issues on phycotoxins are faced to prevent human intoxications: setting up sensitive methods for their detection and providing key information on their toxicity. If analytical chemistry has been largely developed to deal with phycotoxins quantification in seafood, cell-based assays are actually popping up again for targeted and nontargeted toxin detection and will benefit of high-throughput methods for cell culturing and cell response analysis. Simultaneously, ethical concern and new paradigm in toxicology emphasize in vitro toxicity testing for delivering knowledge on key events and pathways involved in cellular response. Hazard assessment also requires information on the behaviour of the toxin within the organism and in vitro assays can provide crucial kinetics data for potential modelling. As cell culture facilities are also evolving with increasing 3D models for mimicking the cell environment in vivo, it is likely that the study of phycotoxins will take advantage of the tremendous progress in cell biology.

Synthetic amorphous silica (SAS) in its nanosized form is now used in food applications although the potential risks for human health have not been evaluated. In this study, genotoxicity and oxidative DNA damage of two pyrogenic (NM-202 and 203) and two precipitated (NM-200 and -201) nanosized SAS were investigated in vivo in rats following oral exposure. Male Sprague Dawley rats were exposed to 5, 10, or 20 mg/kg b.w./day for three days by gavage. DNA strand breaks and oxidative DNA damage were investigated in seven tissues (blood, bone marrow from femur, liver, spleen, kidney, duodenum, and colon) with the alkaline and the (Fpg)-modified comet assays, respectively. Concomitantly, chromosomal damage was investigated in bone marrow and in colon with the micronucleus assay. Additionally, malondialdehyde (MDA), a lipid peroxidation marker, was measured in plasma. When required, a histopathological examination was also conducted. The results showed neither obvious DNA strand breaks nor oxidative damage with the comet assay, irrespective of the dose and the organ investigated. Similarly, no increases in chromosome damage in bone marrow or lipid peroxidation in plasma were detected. However, although the response was not dose-dependent, a weak increase in the percentage of micronucleated cells was observed in the colon of rats treated with the two pyrogenic SAS at the lowest dose (5 mg/kg b.w./day). Additional data are required to confirm this result, considering in particular, the role of agglomeration/aggregation of SAS NMs in their uptake by intestinal cells.

Titanium dioxide (TiO2) nanomaterials (NMs) are widely used in a diversity of products including cosmetics, pharmaceuticals, food, and inks, despite uncertainties surrounding the potential health risks that they pose to humans and the environment. Previous studies on the genotoxicity of TiO2 have reported discrepant or inconclusive findings in both in vitro and in vivo systems. This study explores the in vivo genotoxic potential of a well-characterized uncoated TiO2 NM with an average diameter of 22 nm (NM-102, from JRC repository) using several genotoxicity endpoints in the LacZ plasmid-based transgenic mouse model. Mice were exposed by intravenous injection to two daily doses of NM-102: 10 and 15 mg/kg of body weight/day. Micronuclei were analyzed in peripheral blood reticulocytes 42 hr after the last treatment. DNA strand breaks (comet assay) and gene mutations were determined in the spleens and livers of the same animals 28 days after the last treatment. Histopathological and cytological analyses were also performed in liver samples. Genotoxic effects were not detected in mice exposed to the nanosized TiO2 under the experimental conditions used, despite a moderate inflammatory response that was observed in the liver. Considering the biopersistence of TiO2 in mouse liver and the moderate inflammatory response, the possibility of a secondary genotoxic effect at higher doses and in conditions that result in a stronger inflammatory response, for example, within a longer time window, should be investigated further. Environ. Mol. Mutagen. 55:500–509, 2014. © 2014 Wiley Periodicals, Inc.

International audience; Useful 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.

While MC-LR and MC-RR share significant structural similarity, MC-RR is less cytotoxic than MC-LR. In the current study, we have compared the effects of MC-LR and MC-RR in Caco-2 cells by evaluating cytotoxicity, oxidative stress (reactive oxygen species production), and the cellular proinflammatory response (IL-6 and IL-8 production). Following treatment with 100 µM microcystins (MC), cytotoxicity was two-fold greater with MC-LR as compared to MC-RR after 24 h exposure. Whereas the reactive oxygen species production and IL-6 secretion were similar following a 24-h treatment with either MC, 100 µM MC-LR induced a five-fold greater IL-8 secretion when compared to MC-RR. Our study has demonstrated that, although both MC-LR and MC-RR induced some cytotoxicity in human intestinal cells, a major difference in IL-8 production was observed between the two variants.

This report presents the extensive literature search conducted on 1) the occurrence of different cyanotoxins in food matrices; 2) the analytical methods for their detection; 3) their toxicological profile; 3) the environmental factors affecting toxicity of cyanobacterial population and 4) the combined effects of mixtures of cyanotoxins and other chemicals. It also includes a review of guidelines values or health-alert levels for cyanotoxins in food (or drinking water) adopted worldwide. The methodological aspects and the queries used in the extensive literature search, the collection and screening of retrieved papers and the inventory are briefly described in the report; all details are available in 3 supplementary appendices to this report. The analysis of collected papers indicated that most of them are focused on a single microcystin (MC) variant (MC-LR) out of the almost 100 MC known. Many studies on occurrence are affected by limited quality, due to analytical drawbacks in the detection methods and were not considered in the exposure assessment. Toxicity studies useful for the derivation of health based reference values are few, being many of them carried out using i.p. injection, which is poorly representative of actual human exposure. In addition, those toxicological studies carried out with poorly characterised cyanobacterial extracts or focused on single parameters, using a single dose, devoted to elucidation of mechanism of action, reporting qualitative description of effects were not used for data extraction. The relevant exposure scenarios are also described, although being the available data on exposure very limited, no definite conclusion on the health risks for the exposed population could be drawn. However, the possibility of risky exposure is evidenced for fish and shellfish consumers and for blue-green algae supplements (BGAS) as well in relation to MC contamination. Finally, many data gaps were identified.

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.

Microcystins (MCs) are cyclic hepatotoxins produced by various species of cyanobacteria. Their structure includes two variable amino acids (AA) giving rise to more than 90 MC variants, however most of the studies to date have focused on the most toxic variant: microcystin LR (MC-LR). Ingestion is the major route of human exposure to MCs and several in vivo studies have demonstrated macroscopic effects on the gastro-intestinal tract. However, little information exists concerning the pathways affected by MC variants on intestinal cells. In the current study, we have investigated the effects of MC-RR and MC-LR on the human intestinal cell line Caco-2 using a non-selective method and compared their response at the pangenomic scale. The cells were incubated for 4h or 24h with a range of non-toxic concentrations of MC-RR or MC-LR. Minimal effects were observed after short term exposures (4h) to either MC variant. In contrast, dose dependent modulations of gene transcription levels were observed with MC-RR and MC-LR after 24h. The transcriptomic profiles induced by MC-RR were quite similar to those induced by MC-LR, suggestive of a largely common mechanism of toxicity. However, changes in total gene expression were more pronounced following exposure to MC-LR compared to MC-RR, as revealed by functional annotation. MC-LR affected two principal pathways, the oxidative stress response and cell cycle regulation, which did not elicit significant alteration following MC-RR exposure. This work is the first comparative description of the effects of MC-LR and MC-RR in a human intestinal cell model at the pangenomic scale. It has allowed us to propose differences in the mechanism of toxicity for MC-RR and MC-LR. These results illustrate that taking into account the toxicity of MC variants remains a key point for risk assessment.

DNA adduct formation of the aromatic amine, 4-aminobiphenyl (4-ABP), a known human carcinogen present in tobacco smoke, and the heterocyclic aromatic amines (HAAs), 2-amino-9H-pyrido[2,3-b]indole (AαC), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), and 2-amino-3,8-dimethylmidazo[4,5-f]quinoxaline (MeIQx), potential human carcinogens, which are also present in tobacco smoke or formed during the high-temperature cooking of meats, was investigated in freshly cultured human hepatocytes. The carcinogens (10 μM) were incubated with hepatocytes derived from eight different donors for time periods up to 24 h. The DNA adducts were quantified by liquid chromatography-electrospray ionization mass spectrometry with a linear quadrupole ion trap mass spectrometer. The principal DNA adducts formed for all of the carcinogens were N-(deoxyguanosin-8-yl) (dG-C8) adducts. The levels of adducts ranged from 3.4 to 140 adducts per 10(7) DNA bases. The highest level of adduct formation occurred with AαC, followed by 4-ABP, then by PhIP, MeIQx, and IQ. Human hepatocytes formed dG-C8-HAA-adducts at levels that were up to 100-fold greater than the amounts of adducts produced in rat hepatocytes. In contrast to HAA adducts, the levels of dG-C8-4-ABP adduct formation were similar in human and rat hepatocytes. These DNA binding data demonstrate that the rat, an animal model that is used for carcinogenesis bioassays, significantly underestimates the potential hepatic genotoxicity of HAAs in humans. The high level of DNA adducts formed by AαC, a carcinogen produced in tobacco smoke at levels that are up to 100-fold higher than the amounts of 4-ABP, is noteworthy. The possible causal role of AαC in tobacco-associated cancers warrants investigation.

In temperate countries, the occurrence of cyanobacterial blooms threatens drinking water resources. Consequently, cyanotoxins are increasingly considered in water treatment, and their reactions with chlorine used to disinfect drinking water are particularly investigated. This study presents new elements for further understanding of cylindrospermopsin chlorination, through reactants and by-products monitoring, UV spectrum examination, and cytotoxicity assessment on human intestinal Caco-2 cells. On the one hand, the evolution of mixture UV spectrum indicated that cylindrospermopsin was quickly transformed at least into one intermediate by-product. While mass spectrometry experiments confirmed that cylindrospermopsin was almost totally transformed within 5 min, chlorine was consumed up to 20 min after the beginning of the reaction with a rate of 5 mol per mol of toxin. Then, LC-MS analysis gave rise to the formation of a third cylindrospermopsin by-product in addition to 5-chloro-cylindrospermopsin and cylindrospermopsic acid previously identified. Thanks to the accurate mass measurement provided by the LTQ-Orbitrap mass spectrometer, this new and stable chlorination by-product was assigned the chemical formula C(13)H(18)N(4)O(7)S. On the other hand, both of the mitochondrial and lysosomal activities measured on Caco-2 cells revealed that cylindrospermopsin chlorination significantly decreases mixture cytotoxicity.

Microcystin-LR (MC-LR) is a toxin produced by freshwater cyanobacteria and is a potential threat to human health. MC-LR has been shown to be both a specific inhibitor of serine/threonine protein phosphatases PP1 and PP2A and a potent tumor promoter in rat liver. However, the genotoxic potential of MCs remains unclear. In this article, we investigated the ability of MC-LR to induce DNA damage on rat hepatocytes following intravenous (iv) administration by using two in vivo genotoxicity assays: the unscheduled DNA synthesis (UDS) and the comet assays. The UDS assay measures DNA synthesis induced from the excision repair of DNA damaged regions and the comet assay is a very sensitive technique for detecting various forms of DNA damage. After an exposure time of 2-4 h or 12-16 h and a dose ranging from 12.5 to 50 microg/kg bw, no DNA damage could be observed in both assays on rat hepatocytes following iv administration. These findings have been discussed and compared with recently published genotoxic results obtained in other organs from mice after oral and intraperitoneal treatments to better understand the mechanism of action of this toxin in relation with its cancerogenicity potential.

Microcystin-LR (MC-LR), involved in human and animal poisonings by cyanobacteria, has been shown to be both a potent tumour promoter in rat liver and an inhibitor of serine/threonine protein phosphatases, specifically PP1 and PP2A. The research on the genotoxic potential of MC-LR counts only few in vivo studies. In order to determine the target organs for DNA-damage induction by MC-LR, the single-cell gel electrophoresis (SCGE) or comet assay was performed in mice. Following a single oral administration of 2 and 4mg/kg bw of MC-LR, a statistically significant induction of DNA damage in blood cells was obtained after 3h. However, after an intra-peritoneal injection (ip), DNA lesions were mainly induced in the liver, but were also reported in the kidney, the intestine and the colon. The sensitivity of the ip route compared to the oral route suggested a difference in the bio-disponibility of the toxin. In any case, DNA damage was induced by MC-LR irrespective of the administration route. Among the target organs, the DNA damage induced in the intestinal tissues (ileum and colon) may contribute to an increased cancer risk.

International audience; No evidence for phase I metabolites of the cyanotoxin cylindrospermopsin (CYN) was given using HepaRG cells and different liver tissue fractions when studying metabolic conversion. Although the application of ketoconazole, a CYP3A4 inhibitor, led to a decreased cytotoxicity of CYN, no metabolites were detected applying high resolution mass spectrometry. Quantification of non-modified CYN led to recovery rates of almost 100%. Consequently, reduction of CYN toxicity in the presence of metabolism inhibiting agents must be attributed to alternative pathways.

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.

Okadaic acid (OA) is the main marine toxin implicated in the diarrhetic shellfish poisoning (DSP) in humans after consumption of contaminated bivalve molluscs. We have previously shown that OA was an in vitro aneugenic compound that induced chromosome loss via micronuclei formation in CHO-K1 cells. The aims of this study were to investigate the chromosomal non-disjunction (ND) potential of OA in human lymphocytes and the pathways involved for aneuploidy in CHO-K1 cells. Firstly, we analysed the formation of micronuclei and the non-disjunction for chromosomes 1 and 17 in binucleated human lymphocytes cells with the cytokinesis-blocked micronucleus (CBMN) assay coupled to a fluorescent in situ hybridization (FISH) technique with centromere-specific DNA probes. We showed that OA statistically increased the frequency of micronucleated lymphocytes in the dose range from 20 to 35 nM. However, FISH analysis did not reveal any increase in the non-disjunction for both chromosomes whatever the concentration between 2.5 and 35 nM. However, a significant increase in ND for the chromosome 17 was found at 1 nM. Secondly, in CHO-K1 cells, we investigated the dose and time dependent effects of OA: (i) on cell cycle progression, (ii) on mitotic-phase arrest and (ii) on mitotic spindle and centrosome abnormalities. The results showed that OA induced a progressive accumulation of mitotic CHO-K1 cells in prometaphase, an induction of multipolar mitotic spindle with centrosome amplification and the formation of multinucleated cells. We concluded that OA did not induce chromosome non-disjunction but should more likely induced chromosome loss in human lymphocytes. Moreover, our results obtained in CHO-K1 suggest that OA induced aneuploidy by preventing the chromosome attachment to the mitotic spindle and by amplifying the centrosome. The mode of action of the toxin in relation to its inhibition of protein phosphatases 1 (PP1) and 2A (PP2A) and the mitosis process is discussed.

Okadaic acid (OA), a major polyether toxin involved in diarrhetic shellfish poisoning (DSP), is a potent tumor promoter in rodent skin and glandular stomach and a specific inhibitor of the serine/threonine protein phosphatases PP1 and PP2A. A previous study, which used the cytokinesis-block micronucleus (CBMN) assay in CHO-K1 cells, showed that OA induced chromosome damage in the presence of a rat liver metabolic activation system (S9). To support OA biotransformation by S9, the same test system was performed, and DNA damage induced by OA was measured with and without metabolic activation as well as in the presence of heat-inactivated S9 fraction. The results showed that only in the presence of active S9 did OA significantly increased the frequency of micronucleated binucleated (MNBN) cells. After a 4-h treatment a 2- to 5-fold increase of MNBN cells was observed at 30 nM and at 50 nM of OA. However, without S9 or in the presence of heat-inactivated S9, OA did not induce any chromosome damage. We concluded that OA can be metabolically activated in vitro into metabolites that are more genotoxic. The CBMN assay coupled with fluorescence in situ hybridization (FISH) using a DNA probe for centromere detection was performed to discriminate between clastogenic (chromosome breakage) and aneugenic (chromosome loss) effects. FISH analysis showed that OA metabolites increased in a dose-dependent manner in centromere positive micronuclei (CEN+): 60% of CEN+ at 30 nM and 75% of CEN+ at 50 nM of OA. The uptake of OA into CHO-K1 cells and the biotransformation of the toxin are discussed.

Cylindrospermopsin has been involved in some cyanobacterial blooms associated with animal and human intoxications in different countries. Liver is the main target organ even though thymus and kidney are also affected. Its toxic effect has been shown to be induced by protein synthesis inhibition. However, further research about its toxicological potential is required, as revealed by the U.S. Unregulated Contaminant Monitoring Regulation (US UCMR) meeting in 2001. Induction of DNA damage by cylindrospermopsin has been reported by some authors either by a direct effect on DNA or by an indirect effect on associated macromolecules. This study focused on evaluating its in vitro genotoxic potential using the comet assay coupled to various cell alteration measurements. No DNA damage was detected by the alkaline comet assay on Chinese hamster ovary (CHO) K1 cells after 24 h of treatment with cylindrospermopsin concentrations of 0.5 and 1 μg mL−1. However, inhibition of cell growth was noticed as well as cell blebbing and rounding. These morphological effects were linked to cytoskeletal reorganization (mainly microfilaments) but not to apoptosis. This study concluded that cylindrospermopsin does not obviously react directly with DNA in CHO K1 cells. But the hypothesis of its metabolization into a genotoxic product must be explored further. © 2003 Wiley Periodicals, Inc. Environ Toxicol 18: 353–359, 2003.

Synthetic amorphous silica nanomaterials (SAS) are extensively used in food and tire industries. In many industrial processes, SAS may become aerosolized and lead to occupational exposure of workers through inhalation in particular. However, little is known about the in vivo genotoxicity of these particulate materials. To gain insight into the toxicological properties of four SAS (NM-200, NM-201, NM-202, and NM-203), rats are treated with three consecutive intratracheal instillations of 3, 6, or 12 mg/kg of SAS at 48, 24, and 3 hrs prior to tissue collection (cumulative doses of 9, 18, and 36 mg/kg). Deoxyribonucleic acid (DNA) damage was assessed using erythrocyte micronucleus test and the standard and Fpg-modified comet assays on cells from bronchoalveolar lavage fluid (BALF), lung, blood, spleen, liver, bone marrow, and kidney. Although all of the SAS caused increased dose-dependent changes in lung inflammation as demonstrated by BALF neutrophilia, they did not induce any significant DNA damage. As the amount of SAS reaching the blood stream and subsequently the internal organs is probably to be low following intratracheal instillation, an additional experiment was performed with NM-203. Rats received three consecutive intravenous injections of 5, 10, or 20 mg/kg of SAS at 48, 24, and 3 hrs prior to tissue collection. Despite the hepatotoxicity, thrombocytopenia, and even animal death induced by this nanomaterial, no significant increase in DNA damage or micronucleus frequency was observed in SAS-exposed animals. It was concluded that under experimental conditions, SAS induced obvious toxic effects but did cause any genotoxicity following intratracheal instillation and intravenous injection.

Synthetic amorphous silica (SAS) in its nanosized form is now used in food applications although the potential risks for human health have not been evaluated. In this study, genotoxicity and oxidative DNA damage of two pyrogenic (NM-202 and 203) and two precipitated (NM-200 and -201) nanosized SAS were investigated in vivo in rats following oral exposure. Male Sprague Dawley rats were exposed to 5, 10, or 20 mg/kg b.w./day for three days by gavage. DNA strand breaks and oxidative DNA damage were investigated in seven tissues (blood, bone marrow from femur, liver, spleen, kidney, duodenum, and colon) with the alkaline and the (Fpg)-modified comet assays, respectively. Concomitantly, chromosomal damage was investigated in bone marrow and in colon with the micronucleus assay. Additionally, malondialdehyde (MDA), a lipid peroxidation marker, was measured in plasma. When required, a histopathological examination was also conducted. The results showed neither obvious DNA strand breaks nor oxidative damage with the comet assay, irrespective of the dose and the organ investigated. Similarly, no increases in chromosome damage in bone marrow or lipid peroxidation in plasma were detected. However, although the response was not dose-dependent, a weak increase in the percentage of micronucleated cells was observed in the colon of rats treated with the two pyrogenic SAS at the lowest dose (5 mg/kg b.w./day). Additional data are required to confirm this result, considering in particular, the role of agglomeration/aggregation of SAS NMs in their uptake by intestinal cells.

International audience; Microcystins (MCs) are toxins produced by several cyanobacteria species found worldwide. MC-LR is the most frequent. Here, we used the human Caco-2 cell line grown on semi-permeable filter supports as an in vitro model for determining MC-LR intestinal bidirectional transport. In this study, there was very low and time-dependent apparent permeability of MC-LR. To identify the limiting factors involved in the low permeability of MC-LR, a mathematical model was constructed to get physiologically relevant and informative parameters. The apical-to-basolateral transport was characterised by a rapid and substantial decrease in apical MC-LR concentrations (24–40% of the initial amount). In the basolateral compartment, the concentrations increased slowly after a lag time, but represented only a small fraction of the loaded concentrations (0.3–1.3%) after 24 h. This weak permeability was mainly due to a low clearance of efflux (from the cellular to the basolateral compartment) and effective secretion (from the cellular to the apical compartment). During the basolateral-to-apical transport, we observed a slow decrease in basolateral concentrations and a rapid increase in apical concentrations. In conclusion, modelling has the potential to highlight the key mechanisms involved in the complex kinetics of toxin transport.

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.

Genetic toxicity information is critical for the safety assessment of all xenobiotics. In the absence of carcinogenicity data, genetic toxicity studies may be used to draw conclusions about the carcinogenicity potential of chemicals. However, current in vitro assays have many limitations as they produce a high rate of irrelevant positive data and possible false negative data due to the weakness of the in vitro models used. Based on the knowledge that the majority of human genotoxic carcinogens require metabolic activation to become genotoxic, it is necessary to develop in vitro cell models that mimic human liver metabolism to replace the use of liver S9 fraction, which, though helpful for predicting the potential carcinogenicity of chemicals in rodents, is questionable in humans. We therefore investigate whether the recently described human hepatoma HepaRG cells, which express the major characteristics of liver functions similarly to primary human hepatocytes, could be a suitable model for human genotoxicity assessment. We determine the performance of comet and micronucleus assays in HepaRG cells to predict in vivo genotoxins based on the list of compounds published by European Centre for the Validation of Alternative Methods (ECVAM). Twenty compounds were tested in HepaRG cells with comet and micronucleus assays over a 24-h period. The specificity, the sensitivity, and the accuracy of the two tests were determined. We found that the comet assay had higher specificity (100%) than the micronucleus (MN) test (80%), whereas the latter was far more sensitive (73%) than the former (44%), resulting nonetheless in an accuracy of 72% for the comet assay and 75% for the MN test. Taken together, our data suggest that the HepaRG cell line can be of use in genetic toxicology and that efforts to develop competent human liver cell models should be increased.

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.

The alkaline single cell gel electrophoresis (comet) assay was used to assess in vitro and in vivo genotoxicity of etoposide, a topoisomerase II inhibitor known to induce DNA strand breaks, and chlorothalonil, a fungicide widely used in agriculture. For in vivo studies, rats were sacrificed at various times after treatment and the induction of DNA strand breaks was assessed in whole blood, bone marrow, thymus, liver, kidney cortex and in the distal part of the intestine. One hour after injection, etoposide induced DNA damage in all organs studied except kidney, especially in bone marrow, thymus (presence of HDC) and whole blood. As observed during in vitro comet assay on Chinese hamster ovary (CHO) cells, dose- and time-dependent DNA effects occurred in vivo with a complete disappearance of damage 24 h after administration. Even though apoptotic cells were detected in vitro 48 h after cell exposure to etoposide, such a result was not found in vivo. After chlorothalonil treatment, no DNA strand breaks were observed in rat organs whereas a clear dose-related DNA damage was observed in vitro. The discrepancy between in vivo and in vitro models could be explained by metabolic and mechanistic reasons. Our results show that the in vivo comet assay is able to detect the target organs of etoposide and suggest that chlorothalonil is devoid of appreciable in vivo genotoxic activity under the protocol used.

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.

Among the different tested endpoints, Al- and Ticontaining nanomaterials did notshowany toxicity in intestinal cell lines in vitro. Nevertheless, this absence of effect was not due to an absence of exposure, since particle-specific uptake was reported. Metal particle uptake over a long time period might therefore be relevant for risk assessment of aluminum- and titanium-containing food products.

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.

Consumers may be simultaneously exposed to several pesticide residues in their diet. A previous study identified the seven most common pesticide mixtures to which the French population was exposed through food consumption in 2006. The aim of this study was to investigate if the seven mixtures are potentially cytotoxic and genotoxic and if so, whether compounds in a same mixture have a combined effect. The cytotoxicity and genotoxicity of the seven mixtures were investigated with a new assay (γ-H2AX) using four human cell lines (ACHN, SH-SY5Y, LS-174T, and HepG2). Mixtures were tested at equimolar concentrations and also at concentrations reflecting their actual proportion in the diet. Irrespective of the cell line tested, parallel cytotoxicity of the seven mixtures was observed. Only one mixture was genotoxic for the HepG2 cells at concentrations = 3 μM in equimolar proportion and at 30 μM in actual proportion. Caspase 3/7 activity, the comet assay, and reactive oxygen species production were also investigated using the same mixture and HepG2 cells. Our results suggest that pesticide metabolites from the mixture generated by HepG2 cells were responsible for the observed damage to DNA. Among the five compounds in the genotoxic mixture, only fludioxonil and cyprodinil were genotoxic for HepG2 cells alone at concentrations = 4 and 20 μM, respectively. Our data suggest a combined genotoxic effect of the mixture at low concentrations with a significantly higher effect of the mixture of pesticides than would be expected from the response to the individual compounds. Environ. Mol. Mutagen. 2012. © 2012 Wiley Periodicals, Inc.

International audience