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International audience; BACKGROUND & AIMS: NADPH oxidase 1 (NOX1) has emerged as a prime regulator of intestinal mucosa immunity and homeostasis. Dysregulation of NOX1 may cause inflammatory bowel disease (IBD). It is not clear how NOX1 is regulated in vivo under inflammatory conditions. We studied the role of CK2 in this process.METHODS: The NOX1 organizer subunit, NADPH oxidase organizer 1 (NOXO1), was immunoprecipitated from cytokine-treated colon epithelial cells, and bound proteins were identified by mass spectrometry analysis. Sites on NOXO1 phosphorylated by CK2 were identified by nanoscale liquid chromatography coupled to tandem mass spectrometry. NOX1 activity was determined in colon epithelial cells and colonoids in the presence or absence of CX-4945, a CK2 specific inhibitor. Acute colitis was induced by administration of trinitrobenzenesulfonic acid in mice treated or not with CX-4945. Colon tissues were analyzed by histologic examination, quantitative polymerase chain reaction, and Western blots. CK2 activity, markers of inflammation, and oxidative stress were assessed.RESULTS: We identified CK2 as a major partner of NOXO1 in colon epithelial cells under inflammatory conditions. CK2 directly binds NOXO1 at the C-terminus containing the Phox homology domain and phosphorylates NOXO1 on several sites. CX-4945 increased ROS generation by NOX1 in human colon epithelial cells and organoids. Strikingly, CK2 activity was reduced in trinitrobenzenesulfonic acid-induced acute colitis, and CX-4945 exacerbated colitis inflammation as shown by increased levels of CXCL1, ROS generation, lipid peroxidation, and colon damage.CONCLUSIONS: The ubiquitous protein kinase CK2 limits NOX1 activity via NOXO1 binding and phosphorylation in colonic epithelial cells and lessens experimental colitis. Loss of CK2 activity during acute colitis results in excessive ROS production, contributing to the pathogenesis. Strategies to activate CK2 could be an effective novel therapeutic approach in IBD.

International audience; Mycotoxins and their metabolites are a family of compounds that contains a great diversity of both structure and biological properties. Information on their toxicity is spread within several databases and in scientific literature. Due to the number of molecules and their structure diversity, the cost and time required for hazard evaluation of each compound is unrealistic. In that purpose, new approach methodologies (NAMs) can be applied to evaluate such large set of molecules. Among them, quantitative structure-activity relationship (QSAR) in silico models could be useful to predict the mutagenic and carcinogenic properties of mycotoxins. First, a complete list of 904 mycotoxins and metabolites was built. Then, some known mycotoxins were used to determine the best QSAR tools for mutagenicity and carcinogenicity predictions. The best tool was further applied to the whole list of 904 mycotoxins. At the end, 95 mycotoxins were identified as both mutagen and carcinogen and should be prioritized for further evaluation.

International audience; The genotoxicity of nano-structured synthetic amorphous silica (SAS), a common food additive, was investigated in vivo in rats. A 90-day oral toxicity study was performed according to OECD test guideline 408 and the genotoxicity of pyrogenic SAS nanomaterial NM-203 was assessed in several organs, using complementary tests. Adult Sprague-Dawley rats of both sexes were treated orally for 90 days with 0, 2, 5, 10, 20, or 50 mg SAS/kg bw per day. Dose levels were selected to approximate expected human dietary exposures to SAS. DNA strand breaks were evaluated by the comet assay in blood, bone marrow, liver, and spleen according to OECD test guideline 489; mutations induced in bone marrow precursors of erythrocytes were assessed by the Pig-a assay and chromosome/ genome damage by the micronucleus assay in blood (OECD test guideline 474) and colon. No treatment-related increases of gene (Pig-a) or chromosome/genome (micronucleus) mutations were detected in the blood. The percentage of micronucleated cells was not increased in the colon of treated rats. Among the organs analyzed by the comet assay, the spleen was the only target showing a weak but biologically relevant genotoxic effect.

TiO 2 nanomaterials (NM) have a wide range of industrial applications, including their use in food products. The incorporation of these NMs in consumer products represents a clear concern for public health safety agencies and consumers, and further investigation of the potential impact of these products on human health is necessary. Indeed, since human oral exposure to TiO 2 NMs is expected to increase in the years to come, there exist legitimate concerns about the risk assessment of these nanomaterials present in food products. A considerable amount of studies investigating the adverse effects of TiO 2 NMs have focused on the genotoxic effects of these NMs, and more recently they have been classified by the International Agency for Research on Cancer (IARC) as carcinogen group 2B following inhalation studies (IARC, 2010). While numerous data are available for anatase or mixes of anatase/rutile forms, the toxicity and the genotoxicity of rutile TiO 2 NMs have been rarely investigated. The aim of our study was therefore to investigate the cytotoxic and genotoxic effects of two rutile TiO 2 NMs, differing in surface coating, NM103 (hydrophobic) and NM104 (hydrophilic), on intestinal and hepatic cell models. Following 3 or 24 h treatments with concentrations of TiO 2 NMs from 1.2 to 80 μg/cm 2 , we have assessed the genotoxicity of these NMs with γH2AX, alkaline comet assay and micronucleus (MN) assays. Cellular viability and effects on oxidative stress were also evaluated. Although TEM imaging demonstrated the presence of the two TiO 2 NMs within the cytoplasm, no significant cytotoxic or genotoxic were observed in either cell model. We have also evaluated and taken into account a variety of potential sources of interference of NMs with cellular assays. TiO 2 NMs present in the cytoplasm introduce uncertainty in the scoring of micronuclei, and therefore this assay is not recommended for the evaluation of the genotoxicity of TiO 2 NMs, or other NMs demonstrating similar interference. The unique properties of TiO 2 NMs introduce additional complexity for genotoxicity testing, and caution must be taken in order to obtain reliable results necessary for accurate hazard assessment.

This paper presents the impact on antimicrobial resistance (AMR) in poultry and pig bacteria of the French EcoAntibio plan, a public policy to reduce antimicrobial use in animals. The analysis was performed using sales data of veterinary antimicrobials and AMR data from bacteria obtained at slaughterhouse and from diseased animals. From 2011-2018, fluoroquinolones exposure decreased by 71.5 % for poultry and 89.7 % for pigs. For Campylobacter jejuni isolated from broilers at slaughterhouses, ciprofloxacin resistance increased from 51 % in 2010 to 63 % in 2018, whereas for turkeys the percentages varied from 56 % in 2014 to 63 % in 2018. For commensal E. coli isolated from the caecal content of broilers at slaughterhouses, the resistance to ciprofloxacin - assessed using an epidemiological cut-off value - increased in broiler isolates from 30.7 % in 2010 to 38.1 % in 2018. In turkeys, the percentage of resistant E. coli isolates decreased from 21.3 % in 2014 to 15.2 % in 2018, whereas in pigs, it increased from 1.9 % in 2009 to 5.5 % in 2017. However, for E. coli isolated from diseased animals, when the breakpoints of 2018 were applied, resistance to fluoroquinolones significantly decreased between 2010 and 2018 from 9.0%-5.4% for broilers/hens, from 7.4 % to 3.4 % for turkeys and from 9.4 % to 3.6 % for pigs. These data show that the major, rapid decrease in the exposition to fluoroquinolones had contrasting effects on resistance in the diverse bacterial collections. Co-selection or fitness of resistant strains may explain why changes in AMR do not always closely mirror changes in use.

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International audience

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.

Improving current in vitro genotoxicity tests is an ongoing task for genetic toxicologists. Further, the question on how to deal with positive in vitro results that are demonstrated to not predict genotoxicity or carcinogenicity potential in rodents or humans is a challenge. These two aspects were addressed at the 5th International Workshop on Genotoxicity Testing (IWGT) held in Basel, Switzerland, on August 17-19, 2009. The objectives of the working group (WG) were to make recommendations on the use of cell types or lines, if possible, and to provide evaluations of promising new approaches. Results obtained in rodent cell lines with impaired p53 function (L5178Y, V79, CHL and CHO cells) and human p53-competent cells (peripheral blood lymphocytes, TK6 and HepG2 cells) suggest that a reduction in the percentage of non-relevant positive results for carcinogenicity prediction can be achieved by careful selection of cells used without decreasing the sensitivity of the assays. Therefore, the WG suggested using p53- competent - preferably human - cells in in vitro micronucleus or chromosomal aberration tests. The use of the hepatoma cell line HepaRG for genotoxicity testing was considered promising since these cells possess better phase I and II metabolizing potential compared to cell lines commonly used in this area and may overcome the need for the addition of S9. For dermally applied compounds, the WG agreed that in vitro reconstructed skin models, once validated, will be useful to follow up on positive results from standard in vitro assays as they resemble the properties of human skin (barrier function, metabolism). While the reconstructed skin micronucleus assay has been shown to be further advanced, there was also consensus that the Comet assay should be further evaluated due to its independence from cell proliferation and coverage of a wider spectrum of DNA damage.

A method is described for the identification and the quantitative determination of the triphenylmethane dyes, malachite green (MG), crystal violet (CV), brilliant green (BG) and leuco malachite green (LMG) and leuco crystal violet (LCV). The analytes were isolated from the matrix by liquid-liquid extraction with acetonitrile. Determination was performed using LC-MS/MS with positive electrospray ionisation. 4 different deuterated internal standards were introduced to improve the quantitative performance of the method. The method has been validated in line with the EU criteria of Commission Decision 2002/657/EC in accordance with the minimum required performance limit (MRPL) set at 2 μgkg(-1) for the sum of MG and LMG. For all the monitored compounds, accuracy, intra-day and inter-day precision were determined at each level of fortification (0.5, 0.75, 1.0 and 2.0 μgkg(-1)). Decision limits CCα and detection capabilities CCβ were calculated according to the standard ISO 11843-2. A study on the applicability of the method was conducted on various aquacultured species with the aim to assess the matrix effects. The presence of residues of leuco brilliant green in fish has also been confirmed from experimental study performed on trout treated with brilliant green, using LTQ-Orbitrap mass spectrometer.

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.

There are concerns that more extensive application of disinfectants in the food industry could result in increased resistance of bacteria to antibiotics and that therapeutic failure could ensue. This paper highlights the differences in application and mode of action between antibiotics in human or animal medicine and disinfectants in the food industry. It describes the completely different methods used to determine in-use concentrations in the two contexts. It points out that the minimum inhibitory concentration (MIC) is never the concentration at which disinfectants should be applied. It also discusses erroneous conclusions that may be drawn when the failure of therapy or disinfection is attributed to intrinsic properties of the molecules rather than to misuse of antibiotics or disinfectants. The paper suggests that the intended meaning of the word "resistance" be carefully defined in scientific articles with due reference to the measurement mentioned in the abstract and possibly reflected in the title. It also suggests that in matters of disinfection the word "resistance" be preferred when the phenomenon being studied is killing and "tolerance" when it is the adaptation to inhibitory concentrations.