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International audience; Silica (either crystalline or amorphous) is widely used for different applications and its toxicological assessment depends on its characteristics and intended use. As sustained inflammation induced by crystalline silica is at the root of silicosis, investigating the inflammatory effects induced by amorphous silicas and their persistence is needed. For the development of new grades of synthetic amorphous silicas, it is also desirable to be able to understand better the factors underlying potential adverse effects. Therefore, we used an optimized in vitro macrophage system to investigate the effects of amorphous silicas, and their persistence. By using different amorphous silicas, we demonstrated that the main driver for the adverse effects is a low size of the overall particle/agglomerate; the second driver being a low size of the primary particle. We also demonstrated that the effects were transient. By using silicon dosage in cells, we showed that the transient effects are coupled with a decrease of intracellular silicon levels over time after exposure. To further investigate this phenomenon, a mild enzymatic cell lysis allowed us to show that amorphous silicas are degraded in macrophages over time, explaining the decrease in silicon content and thus the transiency of the effects of amorphous silicas on macrophages.

The ECETOC NanoApp was developed to support industry in the registration of sets of nanoforms, as well as regulators in the evaluation of these registration dossiers. The ECETOC NanoApp uses a systematic approach to create and justify sets of similar nanoforms, following the ECHA guidance in a transparent and evidence-based manner. The rational and decision rules behind the ECETOC NanoApp are described in detail in ���Janer, G., R. Landsiedel, and W. Wohlleben. 2021. [Rationale and Decision Rules Behind the ECETOC NanoApp to Support Registration of Sets of Similar Nanoforms within REACH. Nanotoxicology 15 (2): 145���122. https://doi.org/10.1080/17435390.2020.1842933]���. The decision criteria apply to human health and environmental hazards and risks. Here, we focus mostly on human health hazards; the decision rules are applied to a series of case studies, each consisting of real nanoforms: two barium sulfate nanoforms, four colloidal silica nanoforms, eight ceria nanoforms, and four copper phthalocyanine nanoforms. For each of them, we show step by step how the ECETOC NanoApp rules are applied. The cases include nanoforms that are justified as members of the same set of similar nanoforms based on sufficient similarity of their intrinsic properties (Tier 1). They also include other nanoforms with a relatively high (but insufficient) similarity of intrinsic properties; their similarity could be justified by functional properties (Tier 2). The case studies also include nanoforms that are concluded not to belong to the same set of similar nanoforms. These outcomes of the NanoApp were overall consistent (sometimes conservative) with available in vivo data. We also noted that datasets for various nanoforms were limited and use of the NanoApp may require the generation of data relevant to the decision criteria.

Considerable evidence exists that bacteria detect eukaryotic communication molecules and modify their virulence accordingly. In previous studies, it has been demonstrated that the increasingly antibiotic-resistant pathogen Pseudomonas aeruginosa can detect the human hormones brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP) at micromolar concentrations. In response, the bacterium modifies its behavior to adapt to the host physiology, increasing its overall virulence. The possibility of identifying the bacterial sensor for these hormones and interfering with this sensing mechanism offers an exciting opportunity to directly affect the infection process. Here, we show that BNP and CNP strongly decrease P. aeruginosa biofilm formation. Isatin, an antagonist of human natriuretic peptide receptors (NPR), prevents this effect. Furthermore, the human NPR-C receptor agonist cANF4-23 mimics the effects of natriuretic peptides on P. aeruginosa, while sANP, the NPR-A receptor agonist, appears to be weakly active. We show in silico that NPR-C, a preferential CNP receptor, and the P. aeruginosa protein AmiC have similar three-dimensional (3D) structures and that both CNP and isatin bind to AmiC. We demonstrate that CNP acts as an AmiC agonist, enhancing the expression of the ami operon in P. aeruginosa. Binding of CNP and NPR-C agonists to AmiC was confirmed by microscale thermophoresis. Finally, using an amiC mutant strain, we demonstrated that AmiC is essential for CNP effects on biofilm formation. In conclusion, the AmiC bacterial sensor possesses structural and pharmacological profiles similar to those of the human NPR-C receptor and appears to be a bacterial receptor for human hormones that enables P. aeruginosa to modulate biofilm expression., IMPORTANCE The bacterium Pseudomonas aeruginosa is a highly dangerous opportunist pathogen for immunocompromised hosts, especially cystic fibrosis patients. The sites of P. aeruginosa infection are varied, with predominance in the human lung, in which bacteria are in contact with host molecular messengers such as hormones. The C-type natriuretic peptide (CNP), a hormone produced by lung cells, has been described as a bacterial virulence enhancer. In this study, we showed that the CNP hormone counteracts P. aeruginosa biofilm formation and we identified the bacterial protein AmiC as the sensor involved in the CNP effects. We showed that AmiC could bind specifically CNP. These results show for the first time that a human hormone could be sensed by bacteria through a specific protein, which is an ortholog of the human receptor NPR-C. The bacterium would be able to modify its lifestyle by favoring virulence factor production while reducing biofilm formation.

International audience; Although nanodiamonds (NDs) appear as one of the most promising nanocarbon materials available so far for biomedical applications, their risk for human health remains unknown. Our work was aimed at defining the cytotoxicity and genotoxicity of two sets of commercial carboxylated NDs with diameters below 20 and 100 nm, on six human cell lines chosen as representative of potential target organs: HepG2 and Hep3B (liver), Caki-1 and Hek-293 (kidney), HT29 (intestine) and A549 (lung). Cytotoxicity of NDs was assessed by measuring cell impedance (xCELLigence® system) and cell survival/death by flow cytometry while genotoxicity was assessed by γ-H2Ax foci detection, which is considered the most sensitive technique for studying DNA double-strand breaks. To validate and check the sensitivity of the techniques, aminated polystyrene nanobeads were used as positive control in all assays. Cell incorporation of NDs was also studied by flow cytometry and luminescent N–V center photoluminescence (confirmed by Raman microscopy), to ensure that nanoparticles entered the cells. Overall, we show that NDs effectively entered the cells but NDs do not induce any significant cytotoxic or genotoxic effects on the six cell lines up to an exposure dose of 250 µg/mL. Taken together these results strongly support the huge potential of NDs for human nanomedicine but also their potential as negative control in nanotoxicology studies.

Silica mesoporous nanoparticles have been recently selected for a wide range of applications from electronics to medicine due to their intrinsic properties. Among medical applications, drug delivery using SiO(2) nanoparticles by oral route is under study. Major benefits are expected including higher specificity and sensitivity together with side effect reduction. Since literature shows that very complex and unexpected interactions could occur between nanomaterials and biological systems, one critical issue is to control the nanoparticle cytotoxicity/genotoxicity for normal tissues and specially stomach and intestine when oral route is considered. The aim of the work is to study the cytotoxicity and genotoxicity of SiO(2) nanoparticles on HT29 human intestine cell line, using conventional and innovative methodologies, for measuring cell viability and proliferation, global metabolism, genotoxicity, and nanoparticles uptake. Core-dye doped SiO(2) nanoparticles of 25 and 100 nm were specifically synthesized to track nanoparticles incorporation by confocal and video microscopy. Besides conventional approaches (sulforhodamine B, flow cytometry, and γ-H2Ax foci), we have performed a real-time monitoring of cell proliferation using an impedance-based system which ensure no interference between measures and nanoparticles physicochemical characteristics. Overall, our results showed that SiO(2)-25nm and SiO(2)-100nm induced a rather limited cytotoxic and genotoxic effects on HT-29 cells after a 24 h exposure. However, regarding cell viability and genotoxicity, inverse dose-dependent relationships were observed for SiO(2)-100nm nanoparticles. In conclusion, it seems that the higher the dose of SiO(2)-100nm, the lower the cytotoxic/genotoxic effects, data that well illustrate the complexity in identifying and understanding the hazards of nanoparticles for human health.

International audience; The screening of several crude plant extracts from Gabon for their antimicrobial and antitumoral activities showed us interesting results. After chromatography separation and purification of the alkaloid extracts from Isolona hexaloba (Annonaceae) roots, followed by spectral data analysis, we characterized two bisbenzylisoquinoline: curine and guattegaumerine [1,2,3].We then, examined whether these compounds can regulate bacterial stress and growth. Interestingly, both molecules activate the formation of bacterial biofilm. However, none of the two, at concentrations ranging from 10-17 to 10-6 M, showed an effect on Escherichia coli, Bacillus subtilis, or a mucoid strain of Pseudomonas aeruginosa growth.Using the rat hepatocarcinoma cells (HTC-R) that are resistant to anticancer drugs, we tested the effects of curine and guattegaumerine on the efflux of Rhodamine123 from HTC-R, a substrate for P-glycoprotein encoded by mdr1 gene. Curine and guattegaumerine at 10µM were able to reduce Rhodamine123 efflux by 50% and were not verapamil (Rhodamine efflux inhibitor) competitors.The cytotoxic effect of curine and guattegaumerine was also evaluated on HTC-R cell growth. No effect was found for concentrations ranging from 10-8 to 10-5 M. In contrast, at 10-4 M, curine and guattegaumerine inhibited cell proliferation by 90 and 70% respectively. Thus, we concluded that due to their antiproliferative effect, these two compounds are not specific substrates for P-glycoprotein but may present potential inhibitors for cell growth in cancer treatment.

Depuis 2002, le débat sur les risques associés à la thérapie génique est initié suite à l’annonce que deux enfants inclus dans un essai thérapeutique impliquant une thérapie génique ont développé des effets indésirables important. En Janvier 2005, le débat sur les risques reprit suite à l’interruption du protocole sur les enfants bulle du Pr Fischer à l’hôpital Necker de Paris. Nous avons donc étudié le processus impliqué ainsi que la réflexion éthique associée aux décisions d’arrêt de protocole de recherche. Notre travail a été mené par une équipe pluridisciplinaire combinant chercheurs en santé, généticiens et éthiciens. Nous avons étudié la participation des chercheurs, des patients, des institutions officielles, des comités d’éthique ainsi que des associations de patients dans le processus de décision d’interruption d’un protocole de recherche. Nous avons également analysé les critères jugés les plus pertinents dans l’arrêt d’un protocole de recherche. Enfin nous avons analysé le point de vue des personnes directement impliquées dans la thérapie génique au moyen d’un questionnaire. Toutes les personnes contactées ont présenté un poster de recherche au congrès de la Société Européenne de Thérapie Génique. 62 personnes d’autant d’équipes de recherche différentes, de 17 pays, sur les 350 contactés ont répondu. Selon eux, la décision d’arrêt d’un protocole de recherche doit être prise suite à une consultation des chercheurs, des patients, du ministère de tutelle, d’une agence nationale de régulation ou d’un comité d’éthique ; la légitimité étant accordée à des décisions prises en commun par les chercheurs, les patients et les comités d’éthique. Les incidents sérieux et de façon plus surprenante, les incidents moins graves sont jugés comme étant des critères suffisants pour interrompre un essai. Nous avons fini par analyser les conséquences éthiques, telles que balance bénéfice/risque, processus de régulation ou responsabilité, de ces critères sur l’arrêt d’un protocole de recherche., In 2002, the debate on the risks of gene therapy was initiated following the annoucement that two children included in a clinical trial developed serious adverse effects. In January 2005, the debate was reignited following the interruption of the “bubble kids protocol” at the Hôpital Necker in Paris. We have thus investigated the ethical stakes involved in decisions to stop protocols. This work was carried out by a multidisciplinary team combining ethics researchers and geneticists. We studied the specific participation of researchers, patients, official institution, ethics committees and patient associations in the processes that can lead to an interruption of trial.We also analysed the criterion judged most relevant for halting a trial. Finally, we analyzed the perspective of the actors implicated directly in the provision of gene therapy, by means of a questionnaire. All the individuals contacted had presented a scientific poster at the European Society of Gene Therapy. 62 out of 350 persons, from 17 countries, responded to our questionnaire. According to these respondants, decisions to stop a trial should be taken after consultation with researchers, patients, the ministry, national agencies or ethics committees. Legitimacy was accorded to joint decision-making by researchers, patients and committees. Serious incidents, and surprisingly less serious incidents, clearly emerge as criterion for stopping a trial. We conclude by analyzing the ethical consequences, such as risk/benefit ratios, regulatory processes and responsibility, associated with these criterions and decisions to stop a trial.

Publisher Summary A study was conducted to examine whether the decision to interrupt a protocol, deeply dependent on the benefice/risk ratio, especially in the gene therapy field of research where biologists and physicians, were directly related. This study helped to identify various criteria pointed out by the researchers. So, when an unexpected and serious event happened, about two-thirds of the population declared being in favor of the interruption of the protocol. A serious and expected event is defined by the death of a patient or a highly allergic reaction. When multiple, less serious events occur, about 53% of the population is also in favor of an interruption of a protocol, but the correlation between these incidents and the protocol has to be clearly demonstrated. The benefice/risk ratio has to be evaluated before each decision. A codification of the criteria, helping in the evaluation of the benefice/risk ratio could be developed in the future in gene therapy, whose goal is the long-term modification of a gene correcting a nonfunctional molecule. So, when a physician decides to include a person to a protocol or to interrupt a protocol, he needs to keep in mind the possible benefit that other patient could have if the protocol continues. About 90% of the population is in favor of the creation of a multidisciplinary independent European and transnational committee involved and specialized in the regulation of gene therapy. Abstract The decision to interrupt a protocol is deeply dependent on the benefice/risk ratio, especially in the gene therapy field of research where biologists and physicians are directly related. Through the 350 gene therapy researchers selected, because they have presented a research poster during the XIth annual congress of the European Society of Gene Therapy in Edinburgh (November 2003), 62 answered our online anonymous questionnaire. Some important possible axis of regulation criteria and processes were underlined. The online version of this questionnaire is still available on our website: http://sergent.jacques.free.fr . Questioned about three main research axis (criteria suggested, legitimate and proper process to regulate research and the necessity of a moratorium on somatic gene therapy), the gene therapy community has demonstrated the importance of a proper regulation. Suggesting the Jesse Gelsinger case (1999) and the Bubble kids of Necker (2002), they have also demonstrated their wish to see a regulation process involving a committee, particularly because of their transnational and multidisciplinarity qualities. The possible interactions with not only the patients but also the associations of patients were also pointed out. A regulation mediated by the national ministries was the unique process that encountered more than 50% of approval. The necessity to have a European regulation through a committee has been demonstrated. In fact, about 75% of the researchers encourage a European regulation and approve the extension of a decision taken in one European country to the European community, if this decision is scientifically demonstrated for a multi-country shared protocol. Nevertheless, only 50% of the population approve the creation of a European law, which cannot be adapted to each individual decision. The inter-activity, which exists in a committee, is the characteristic mentioned by the population, determinant in a possible European regulation. The unawareness of the actual charters and declarations with interest in gene therapy has also been shown by this study, especially by the directors of laboratories or studies.

Silicon dioxide nanoparticles (n-SiO2) have recently encountered a wide variety of applications in medicine or engineering but their toxicological effects are poorly understood. In this study, we have used SiO2-25 nm and SiO2-100 nm mono-dispersed nanoparticles labeled with Rhodamine B and TMPyP respectively. These two fluorophores were incorporated during synthesis in order to track nanoparticles cell incorporation. Up-to-date, no evaluation of the toxicological effects of these nanoparticles upon human kidney has been published. As kidney is one of the major traditional retention organs, the aim of our study is to evaluate the potential toxicity of these nanoparticles on two human cell lines from proximal tubule (Caki-1 and Hek293). Our results report that the two cell lines do not show similar responses after 24 hours of exposure to SiO2-nanoparticles disregarding a similar origin in the kidney. Interestingly, our results indicate that for both tested SiO2-nanoparticles, Caki-1 cells present a higher sensitivity in terms of cytotoxicity and genotoxicity than Hek293 cells. Furthermore, our results show that for similar concentration of exposure, SiO2-25 nm seems to be more cytotoxic and genotoxic than SiO2-100nm for both tested cell lines.

International audience; Nanomaterials are versatile materials with a vast repertoire of properties and potential applications. It is expected that they will be used in progressively incremental quantities in the future, giving rise to concerns about to their potential impact on living beings and the environment. The science of studying these effects of nanomaterials can be termed as “Nanotoxicology.” Nanotoxicology has evolved to date based on a combination of several in vitro, in silico, and in vivo experimental studies. Epidemiological information on nanomaterials is still lacking, rendering our understanding of these new materials, incomplete. This chapter makes a case for taking this field one step further in terms of combining in vitro, in vivo, and in silico methods to obtain an in-depth understanding of safety aspects associated with broad classes of nanomaterials in a manner never tried before. This combined approach is especially necessary in light of evolving regulations pertaining to nanomaterials, and the possibility of avoiding epidemiological challenges through in silico predictions (also known as computer-based simulations of complex biological phenomena). An overview of approaches to characterize nanomaterials, and the regulations associated with them will also be presented in this chapter.