miR‐4510 blocks hepatocellular carcinoma development through RAF1 targeting and RAS/RAF/MEK/ERK signalling inactivation

International audienceBACKGROUND:Therapeutic outcomes using the multikinase inhibitors, sorafenib and regorafenib, remain unsatisfactory for patients with advanced hepatocellular carcinoma (HCC). Thus, new drug modalities are needed. We recently reported the remarkable capacity of miR-4510 to impede the growth of HCC and hepatoblastoma through Glypican-3 (GPC3) targeting and Wnt pathway inactivation.METHODS:To identify new targets of miR-4510, we used a label-free proteomic approach and reported down-regulation of RAF proto-oncogene serine/threonine-protein kinase (RAF1) by miR-4510. Because the tumourigenic role of RAF1 in HCC is controversial, we further studied RAF1:miR-4510 interactions using cellular, molecular as well as functional approaches and a chicken chorioallantoic membrane (CAM) xenograft model.RESULTS:We found an increase in RAF1 protein in 59.3% of HCC patients and a specific up-regulation of its transcript in proliferative tumours. We showed that miR-4510 inactivates the RAS/RAF/MEK/ERK pathway and reduces the expression of downstream targets (ie c-Fos proto-oncogene [FOS]) through RAF1 direct targeting. At a cellular level, miR-4510 inhibited HCC cell proliferation and migration and induced senescence in part by lowering RAF1 messenger RNA (mRNA) and protein expression. Finally, we confirmed the pro-tumoural function of RAF1 protein in HCC cells and its ability to sustain HCC tumour progression in vitro and in vivo.CONCLUSIONS:In this work, we confirm that RAF1 acts as an oncogene in HCC and further demonstrate that miR-4510 acts as a strong tumour suppressor in the liver by targeting many proto-oncogenes, including GPC3 and RAF1, and subsequently controlling key biological and signalling pathways among which Wnt and RAS/RAF/MEK/ERK signals

Validating intestinal effects of food-grade titanium dioxide using a murine gut organoid model as alternative to in vivo models

International audienceBackground: Nanoparticles (NPs) found in the human diet mainly originate from inorganic food additives, often used quantum satis in common foodstuff, which raises public health concerns due to daily exposure. The whitener and opacifying agent titanium dioxide (TiO2, E171 in EU) is one of the most studied nanomaterial, evoking inflammatory responses and precancerous lesions in the rodent intestine. Investigating the potential hazards of chronic oral exposure to NPs is often time-consuming and requires animal models, specific spaces and skills. However, recent technical advances in stem cells and threedimensional cultures allowed the use of organoids as an alternative model to in vivo experiments. Herein we used murine intestinal organoids to characterize intestinal impacts of food-grade TiO2 in comparison to already reported in vivo data, and to validate organoids as a reliable model for studying the effects of foodborne NPs in the gut.Methods: Three different wild-type C57bl/6 mice were used for small intestine collection. Intestinal crypts were purified, dissociated, and cells were cultured for organoid growth. After 4 passages, organoids were dissociated and seeded as a 2.5D culture, then exposed to 0.1, 1, 10 or 100µg/ml of E171 for 24h. Supernatants were collected, and cytotoxicity assessed by LDH release quantification. Total RNA was extracted from samples and analyzed for cell proliferation and differentiation, genotoxicity, antimicrobial peptides, permeability, oxidative stress, Toll Like Receptors (TLR), NFκB, cytokine and chemokine gene expressions by qPCR. Cell apoptosis was also evaluated by cleaved Caspase-3 quantification using immunofluorescence.Results: Gut organoids exposed to E171 showed a dose-dependent up-regulation of the cell proliferation marker Mki67 together with increased protein expression of cleaved-Caspase-3, suggesting epithelium renewal or restructuring. This occurred in parallel to a decreased expression of the enterocyte differentiation markers Alpi and Krt20 as well as up-regulation of the neuroendocrine marker Chga. Moreover, food-grade E171 decreased gene expression of antimicrobial peptides (Lyz, Reg3b, S100a8) and tight junction proteins (F11r, Tjp1, Ocln, Cldn7, Cldn15), suggesting altered epithelial secretion and permeability. We also showed that the TLR4-NFκB pathway was negatively impacted in a dose-dependent manner, while oxidative stress, cytokine and chemokine gene expressions remained unaltered. Although E171 exposure was not cytotoxic, TiO2 increased expression of gadd45a at low dose (i.e. 1µg/ml), suggesting DNA damage.Conclusions: Taking together, a 24h-exposure of murine intestinal organoids to food-grade TiO2 impacts epithelial barrier integrity (cell proliferation and differentiation, gut permeability, genotoxic effect) and antimicrobial defenses as reported in vivo in rodent models, hence validating the use of intestinal organoids for toxicological studies of foodborne NPs

Prognostic Risk Classification for Biochemical Relapse-Free Survival in Oligometastatic Recurrent Prostate Cancer Determined by Choline PET

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A posteriori dietary patterns better explain variations of the gut microbiome than individual markers in the American Gut Project

BackgroundIndividual diet components and specific dietary regimens have been shown to impact the gut microbiome.ObjectivesHere, we explored the contribution of long-term diet by searching for dietary patterns that would best associate with the gut microbiome in a population-based cohort.MethodsUsing a priori and a posteriori approaches, we constructed dietary patterns from an FFQ completed by 1800 adults in the American Gut Project. Dietary patterns were defined as groups of participants or combinations of food variables (factors) driven by criteria ranging from individual nutrients to overall diet. We associated these patterns with 16S ribosomal RNA-based gut microbiome data for a subset of 744 participants.ResultsCompared to individual features (e.g., fiber and protein), or to factors representing a reduced number of dietary features, 5 a posteriori dietary patterns based on food groups were best associated with gut microbiome beta diversity (P ≤ 0.0002). Two patterns followed Prudent-like diets-Plant-Based and Flexitarian-and exhibited the highest Healthy Eating Index 2010 (HEI-2010) scores. Two other patterns presented Western-like diets with a gradient in HEI-2010 scores. A fifth pattern consisted mostly of participants following an Exclusion diet (e.g., low carbohydrate). Notably, gut microbiome alpha diversity was significantly lower in the most Western pattern compared to the Flexitarian pattern (P ≤ 0.009), and the Exclusion diet pattern was associated with low relative abundance of Bifidobacterium (P ≤ 1.2 × 10-7), which was better explained by diet than health status.ConclusionsWe demonstrated that global-diet a posteriori patterns were more associated with gut microbiome variations than individual dietary features among adults in the United States. These results confirm that evaluating diet as a whole is important when studying the gut microbiome. It will also facilitate the design of more personalized dietary strategies in general populations

Human TR146 cells and pig buccal mucosa to assess oral transmucosal passage and buccal toxicity of foodgrade titanium dioxide

International audienceBackground: Today, the use of titanium dioxide (TiO 2) as food additive (E171) has been banned by the European Commission, due to concerns for human health based on studies showing TiO 2 particles systematically available, tissue accumulation, a genotoxic risk and possible promotion of precancerous lesions. However, E171 is still present in toothpastes and pharmaceutical tablets as a whitening powder mixing nano-and submicronic particles. Risk assessment of TiO 2 intake by oral route is mainly based on the assumption that particles are mainly absorbed by the intestine. However, while the buccal mucosa is the first exposed area, the possibility of an oro-transmucosal passage has not been documented so far. In order to gain insight on possible adverse effects for human health associated to E171 buccal exposure, we analyzed in vivo the translocation of TiO 2 (E171) in the buccal mucosa of pigs used as human mouth model. Moreover, we evaluated in vitro the particle translocation on human buccal TR146 cell line, and measured cytotoxic and genotoxic effects on proliferative and differentiated epithelial cells. Methods & Results: Under realistic exposure conditions with 50 µg/ml of food-grade TiO 2 in water suspension (size distribution 20-440 nm; mean size of 105 nm) deposited under the tongue of pigs, TEM-EDX data revealed the presence of small aggregates of TiO 2 particles translocated into the buccal mucosa from 30 minutes of exposure, reaching submaxillary lymph nodes after 4 hours. In human TR146 cells exposed to E171, kinetic analysis using confocal, TEM and SIMS imaging showed progressive and large uptake of isolated or small aggregates of both submicronic and nanosized particles, showing high permeability capacity. At 2h of E171 exposure, cytotoxicity, genotoxicity and oxidative stress were investigated on both proliferative or differentiated TR146 cells, in comparison with two TiO 2 size standards of 115 nm and 21 nm in diameter. All tested TiO 2 particles were reported cytotoxic on proliferative TR146 cells, and this effect was almost abolished following differentiation. Oxidative stress and genotoxicity assessed through γH2AX and 53BP1 foci formation and comet assay were only reported for E171 sample and TiO 2 particles of 115 nm, suggesting the particles above 20 nm responsive of these effects, and mainly on proliferative cells. Conclusions: Taken together, these results show in vivo and in vitro that the buccal mucosa is an important absorption route for systemic passage of foodborne TiO 2 (E171) particles. In human cells, uptake of TiO2 particles was cytotoxic without size effects, while they generate further oxidative and genotoxic stresses in proliferative buccal cells, that could impair epithelium renewal in the mouth. Altogether, these data emphasize that buccal exposure should be considered for toxicokinetic and risk assessments of TiO2 in human when used as food additive, including in toothpaste and pharmaceutical formulations

Human TR146 cells and pig buccal mucosa to assess oral transmucosal passage and buccal toxicity of foodgrade titanium dioxide

International audienceBackground: Today, the use of titanium dioxide (TiO 2) as food additive (E171) has been banned by the European Commission, due to concerns for human health based on studies showing TiO 2 particles systematically available, tissue accumulation, a genotoxic risk and possible promotion of precancerous lesions. However, E171 is still present in toothpastes and pharmaceutical tablets as a whitening powder mixing nano-and submicronic particles. Risk assessment of TiO 2 intake by oral route is mainly based on the assumption that particles are mainly absorbed by the intestine. However, while the buccal mucosa is the first exposed area, the possibility of an oro-transmucosal passage has not been documented so far. In order to gain insight on possible adverse effects for human health associated to E171 buccal exposure, we analyzed in vivo the translocation of TiO 2 (E171) in the buccal mucosa of pigs used as human mouth model. Moreover, we evaluated in vitro the particle translocation on human buccal TR146 cell line, and measured cytotoxic and genotoxic effects on proliferative and differentiated epithelial cells. Methods & Results: Under realistic exposure conditions with 50 µg/ml of food-grade TiO 2 in water suspension (size distribution 20-440 nm; mean size of 105 nm) deposited under the tongue of pigs, TEM-EDX data revealed the presence of small aggregates of TiO 2 particles translocated into the buccal mucosa from 30 minutes of exposure, reaching submaxillary lymph nodes after 4 hours. In human TR146 cells exposed to E171, kinetic analysis using confocal, TEM and SIMS imaging showed progressive and large uptake of isolated or small aggregates of both submicronic and nanosized particles, showing high permeability capacity. At 2h of E171 exposure, cytotoxicity, genotoxicity and oxidative stress were investigated on both proliferative or differentiated TR146 cells, in comparison with two TiO 2 size standards of 115 nm and 21 nm in diameter. All tested TiO 2 particles were reported cytotoxic on proliferative TR146 cells, and this effect was almost abolished following differentiation. Oxidative stress and genotoxicity assessed through γH2AX and 53BP1 foci formation and comet assay were only reported for E171 sample and TiO 2 particles of 115 nm, suggesting the particles above 20 nm responsive of these effects, and mainly on proliferative cells. Conclusions: Taken together, these results show in vivo and in vitro that the buccal mucosa is an important absorption route for systemic passage of foodborne TiO 2 (E171) particles. In human cells, uptake of TiO2 particles was cytotoxic without size effects, while they generate further oxidative and genotoxic stresses in proliferative buccal cells, that could impair epithelium renewal in the mouth. Altogether, these data emphasize that buccal exposure should be considered for toxicokinetic and risk assessments of TiO2 in human when used as food additive, including in toothpaste and pharmaceutical formulations

Food-grade titanium dioxide translocates across the oral mucosa in pigs and induces genotoxicity in an in vitro model of human oral epithelium

BackgroundFood-grade titanium dioxide (TiO2), composed of nano- and submicron-sized particles, is used worldwide in various foodstuffs, toothpastes and pharmaceutical tablets as a whitening and opacifying agent. Its use as a food additive (E171 in EU) has raised concerns for human health regarding its systemic availability, tissue accumulation, genotoxicity and promotion of precancerous lesions. However, although the buccal mucosa is the first area exposed, oral transmucosal passage of TiO2 particles has not been documented. Here we analyzed TiO2 (E171) particle translocation in vivo through the pig buccal mucosa and in vitro on human buccal TR146 cells, and the effects of E171 on proliferating and differentiated human oral epithelial cells.ResultsUsing transmission electronic microscopy (TEM) coupled to energy-dispersive X-ray spectroscopy (EDX), isolated TiO2 particles and small aggregates were observed in the buccal floor of pigs starting 30 min after the sublingual deposition of E171 suspended in water, and recovered in the submandibular lymph nodes at 4 h. In human TR146 cells exposed to E171, kinetic analyses using confocal microscopy, TEM and high-resolution secondary ion mass spectrometry (SIMS) imaging showed high uptake capacities of both the nano- and submicron-sized TiO2 particles. At 2 h, the cytotoxicity, genotoxicity and oxidative stress were investigated in both proliferating and differentiated TR146 cells exposed to E171 in comparison with two TiO2 size standards of 115 and 21 nm in diameter. All TiO2 samples were reported cytotoxic in proliferating cells, an effect almost abolished following differentiation. Genotoxicity (γH2AX or 53BP1 foci formation and comet assays) and oxidative stress (CellRox reagent) were only reported for the E171 and 115 nm TiO2 particles, and mainly in proliferating cells.ConclusionsThese data showed that the buccal mucosa is an important absorption route for the systemic passage of food-grade TiO2 particles. In human cells, TiO2 particles are cytotoxic and generate size-dependent oxidative and genotoxic stresses in proliferating cells, potentially impairing oral epithelium renewal. Altogether, these data emphasize that buccal exposure should be considered during toxicokinetic studies and for risk assessment of TiO2 in human when used as food additive, including in toothpastes and pharmaceutical formulations

PARP inhibitors and radiation potentiate liver cell death in vitro. Do hepatocellular carcinomas have an achilles’ heel?

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PARP Inhibition Increases the Response to Chemotherapy in Uveal Melanoma

Uveal melanoma (UM) remains without effective therapy at the metastatic stage, which is associated with BAP-1 (BRCA1 associated protein) mutations. However, no data on DNA repair capacities in UM are available. Here, we use UM patient-derived xenografts (PDXs) to study the therapeutic activity of the PARP inhibitor olaparib, alone or in combination. First, we show that the expression and the activity of PARP proteins is similar between the PDXs and the corresponding patient’s tumors. In vivo experiments in the PDX models showed that olaparib was not efficient alone, but significantly increased the efficacy of dacarbazine. Finally, using reverse phase protein arrays and immunohistochemistry, we identified proteins involved in DNA repair and apoptosis as potential biomarkers predicting response to the combination of olaparib and dacarbazine. We also observed a high increase of phosphorylated YAP and TAZ proteins after dacarbazine + olaparib treatment. Our results suggest that PARP inhibition in combination with the alkylating agent dacarbazine could be of clinical interest for UM treatment. We also observe an interesting effect of dacarbazine on the Hippo pathway, confirming the importance of this pathway in UM

Évaluation des risques sanitaires des acides haloacétiques dans l’eau destinée à la consommation humaine

Citation suggérée : Anses. (2023). Évaluation des risques sanitaires des acides haloacétiques dans l’eau destinée à la consommation humaine. (saisine 2021-SA-0015). Maisons-Alfort : Anses, 315 p.Les acides haloacétiques (AHA) sont principalement des sous-produits de désinfection (SPD)issus de réactions chimiques entre le chlore, la matière organique et les ions bromures ouiodures présents dans l’eau. Jusqu’à récemment, les AHA ne faisaient pas partie des paramètres couverts par la réglementation nationale pour les eaux destinées à la consommation humaine (EDCH).Une limite de qualité fixée à 60 μg.L est introduite par la nouvelle directive européenne relative à la qualité des eaux destinées à la consommation humaine du 16 décembre 2020 pour la somme des cinq AHA suivants (AHA5) : les acides monochloroacétique, dichloroacétique, trichloroacétique, monobromoacétique et dibromoacétique (AMCA, ADCA, ATCA, AMBA et ADBA). Cette limite de qualité n’est pas basée sur des critères sanitaires. Elle vise à réduire les concentrations de ces sous-produits de chloration dans les EDCH sans compromettre l’efficacité de la désinfection. En décembre 2022, cette limite de qualité a été intégrée à la réglementation nationale (arrêté du 11 janvier 2007 modifié). Cette limite de qualité est applicable en France depuis le 1er janvier 2023. Toutefois, la mise en œuvre de l’analyse de ces paramètres à travers le contrôle sanitaire des EDCH sera effective au plus tard en janvier 2026.En prévision de l’introduction de ce nouveau paramètre dans la réglementation relative à laqualité des EDCH, la direction générale de la santé (DGS) avait confié au laboratoired’hydrologie de Nancy (LHN) de l’Anses, en 2016 la réalisation d’une campagne nationaleexploratoire dans les eaux brutes et les eaux distribuées au robinet du consommateur portantnotamment sur l’analyse de neuf AHA, comprenant les cinq AHA de la nouvelle directiveeuropéenne ainsi que quatre AHA non réglementés : les acides tribromoacétique,bromochloroacétique, bromodichloroacétique, dibromochloroacétique (ATBA, ABCA, ABDCAet ADBCA). Cette campagne, qui s’est tenue sur la période 2016-2017 a mis en évidence queparmi les cinq AHA réglementés, deux sont majoritaires en termes de fréquence dequantification1 dans les eaux traitées (ADCA et ATCA). L’AMBA et l’AMCA n’ont en revanchejamais été quantifiés que ce soit dans les eaux brutes ou traitées. Concernant les AHA noncouverts par la réglementation européenne, l’ABDCA et l’ADBCA ont été fréquemmentquantifiés dans les eaux traitées.Dans ce contexte, la DGS a saisi l’Anses le 23 décembre 2020 afin de réaliser une évaluationdes risques sanitaires liés à la présence d’AHA dans les EDCH, en tenant particulièrement compte des deux AHA fréquemment quantifiés dans les eaux traitées dans la campagne de l’Anses et qui ne sont pas couverts par la réglementation européenne (ABDCA et ADBCA).Conformément au contrat d’expertise en date du 25 février 2021, suite à une première recherche bibliographique et à une 1ère réunion du GT ERS EDCH sur le sujet, l’Anses a décidé de cibler l’évaluation des risques sanitaires sur les neuf AHA chlorés et bromés dont la présence a été documentée dans les EDCH en France, en particulier à travers la campagne d’analyse 2016-2017 du LHN dans l’EDCH, soit les molécules suivantes :- cinq AHA réglementés (arrêtés du 11 janvier 2007 modifiés) :o acide monochloroacétique (AMCA) ;o acide dichloroacétique (ADCA) ;o acide trichloroacétique (ATCA) ;o acide monobromoacétique (AMBA) ;o acide dibromoacétique (ADBA) ;- quatre AHA non réglementés :o acide tribromoacétique (ATBA) ;o acide bromochloroacétique (ABCA) ;o acide dibromochloroacétique (ADBCA) ;o acide bromodichloroacétique (ABDCA)