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Rodent studies on the effects of engineered nanomaterials (ENM) on the gut microbiome have revealed contradictory results. Our aim was to assess the effects of four well-investigated model ENM using a realistic exposure scenario. Two independent ad libitum feeding studies were performed. In study 1, female mice from the local breeding facility received feed pellets containing 1% CeO2 or 1% SiO2 for three weeks. In study 2, both female and male mice were purchased and exposed to 0.2% Ag-PVP or 1% TiO2 for four weeks. A next generation 16S rDNA sequencing-based approach was applied to assess impacts on the gut microbiome. None of the ENM had an effect on the α- or β-diversity. A decreased relative abundance of the phylum Actinobacteria was observed in SiO2 exposed mice. In female mice, the relative abundance of the genus Roseburia was increased with Ag exposure. Furthermore, in study 2, a sex-related difference in the β-diversity was observed. A difference in the β-diversity was also shown between the female control mice of the two studies. We did not find major effects on the gut microbiome. This contrast to other studies may be due to variations in the study design. Our investigation underlined the important role of the sex of test animals and their microbiome composition prior to ENM exposure initiation. Hence, standardization of microbiome studies is strongly required to increase comparability. The ENM-specific effects on Actinobacteria and Roseburia, two taxa pivotal for the human gut homeostasis, warrant further research on their relevance for health.

Respirable crystalline silica (RCS) is the leading cause of occupational respiratory diseases worldwide. RCS is associated with inflammatory lung reactions, which can lead to silicosis, cancer, and autoimmune diseases. The extreme variability of silica specimens, including its amorphous forms, the surface heterogeneity, and variable toxicity effects, generated one of the most intriguing enigmas in particle toxicology, i.e., deciphering which physico-chemical features explain and predict the variable hazard of silica. Using a set of ad hoc prepared synthetic and natural quartz particles, we have identified a unique subfamily of surface silanols as a key initiator of the toxicity of silica particles. These moieties, namely the "nearly-free silanols" (NFS), appear on the surface of quartz when crystals are fractured, and their amount can be modulated by physico-chemical processes. The peculiar spatial arrangement of NFS was demonstrated as the most energetically favorable and selective for interacting zwitterionic phospholipids that make up cell membranes. The toxic activity of NFS was also confirmed with amorphous nanosilica synthesized at high temperatures, and our recent findings suggest that NFS could impart toxic properties to other silica polymorphs and hydroxylated surfaces, including aluminosilicates and engineered stones. Overall, NFS occurrence accounts for the origin and variability of the toxicity of silica, opening perspectives for controlling RCS hazard, and may contribute to understand other important interfacial phenomena of hydroxylated materials.

Respirable crystalline silica (RCS) is the leading cause of occupational respiratory disease worldwide. RCS is associated with silicosis, cancer, and autoimmune diseases [1]. Silica (SiO2) is a simple, yet structurally very complex oxide and tens of variable crystalline and amorphous forms exist with different structures and surfaces. Structural heterogeneity is reflected in variable toxic effects, and this in turn generates one of the most intriguing enigmas in particle toxicology, i.e., deciphering the exact molecular nature of the interaction between silica and biological matter.A large set of synthetic and natural, crystalline and amorphous, micrometric and nanometric silica particles were prepared, modified, and characterized. The interaction of these surface-modified silicas with membrane systems of decreasing molecular complexity, e.g., red blood cells, liposomes, and phospholipid supramolecular structures (PLS), was investigated and compared with the results of in vitro and in vivo particle toxicity assessment.A specific silanol (≡Si-OH) sub-group at silica surface, the “nearly free silanols” (NFS), was evidenced as the cause for the membranolytic and inflammatory effect of silica [2]. Silica powders with NFS-rich surfaces caused RBC membrane lysis, and selectively perturbed liposomes and adsorbed PLS. Specific amino groups exposed at the membrane surface are proposed as recognition epitopes for the selective interaction with NFS, which are in turn proposed as the molecular pattern that defines the interaction of silica with biomembranes [3]. Our findings open a new perspective for tailoring less toxic silica particles and for designing improved technological applications of silica. NFS and hydroxylated surface moieties may be also relevant for the toxicity of other respirable mineral dusts, suggesting a new paradigm for particle toxicity mechanism.References[1] Leung et al., Lancet 2012, 379, 2008; [2] Pavan et al., Proc. Natl. Acad. Sci USA 2020, 117, 27836; [3] Pavan et al., sumbitted to ACS Central Science

Background Inhalation of multi-walled carbon nanotubes (MWCNTs) poses a potential risk to human health. In order to safeguard workers and consumers, the toxic properties of MWCNTs need to be identified. Functionalization has been shown to either decrease or increase MWCNT-related pulmonary injury, depending on the type of modification. We, therefore, investigated both acute and chronic pulmonary toxicity of a library of MWCNTs derived from a common pristine parent compound (NC7000). Methods MWCNTs were thermally or chemically purified and subsequently surface functionalized by carboxylation or amination. To evaluate pulmonary toxicity, male C57BL6 mice were dosed via oropharyngeal aspiration with either 1.6 or 4 mg/kg of each MWCNT type. Mitsui-7 MWCNT was used as a positive control. Necropsy was performed at days 3 and 60 post-exposure to collect bronchoalveolar lavage fluid (BALF) and lungs. Results At day 3 all MWCNTs increased the number of neutrophils in BALF. Chemical purification had a greater effect on pro-inflammatory cytokines (IL-1β, IL-6, CXCL1) in BALF, while thermal purification had a greater effect on pro-fibrotic cytokines (CCL2, OPN, TGF-β1). At day 60, thermally purified, carboxylated MWCNTs had the strongest effect on lymphocyte numbers in BALF. Thermally purified MWCNTs caused the greatest increase in LDH and total protein in BALF. Furthermore, the thermally purified and carboxyl- or amine-functionalized MWCNTs caused the greatest number of granulomatous lesions in the lungs. The physicochemical characteristics mainly associated with increased toxicity of the thermally purified derivatives were decreased surface defects and decreased amorphous content as indicated by Raman spectroscopy. Conclusions These data demonstrate that the purification method is an important determinant of lung toxicity induced by carboxyl- and amine-functionalized MWCNTs.

Excessive inhalation of cobalt (Co) dust can have harmful effects on the respiratory tract, yet all cobalt substances do not have the same potential for inducing toxicity. The prevalent hypothesis is that the potential of Co substances to release Co

Rodent studies on the effects of engineered nanomaterials (ENM) on the gut microbiome have revealed contradictory results. Our aim was to assess the effects of four well-investigated model ENM using a realistic exposure scenario. Two independent

Macrophages are not only derived from circulating blood monocytes or embryonic precursors but also expand by proliferation. The origin determines macrophage fate and functions in steady state and pathological conditions. Macrophages predominantly infiltrate fibre-induced mesothelioma tumors and contribute to cancer development. Here, we revealed their ontogeny by comparing the response to needle-like mesotheliomagenic carbon nanotubes (CNT-7) with tangled-like non-mesotheliomagenic CNT-T. In a rat peritoneal cavity model of mesothelioma, both CNT induced a rapid macrophage disappearance reaction (MDR) of MHCIIlow resident macrophages generating an empty niche available for macrophage repopulation. Macrophage depletion after mesotheliomagenic CNT-7 was followed by a substantial inflammatory reaction, and macrophage replenishment completed after 7 days. Thirty days after non-mesotheliomagenic CNT-T, macrophage repopulation was still incomplete and accompanied by a limited inflammatory reaction. Cell depletion experiments, flow cytometry and RNA-seq analysis demonstrated that, after mesotheliomagenic CNT-7 exposure, resident macrophages were mainly replaced by an influx of monocytes, which differentiated locally into MHCIIhigh inflammatory macrophages. In contrast, the low inflammatory response induced by CNT-T was associated by the accumulation of self-renewing MHCIIlow macrophages that initially derive from monocytes. In conclusion, the mesotheliomagenic response to CNT specifically relies on macrophage niche recolonization by monocyte-derived inflammatory macrophages. In contrast, the apparent homeostasis after non-mesotheliomagenic CNT treatment involves a macrophage regeneration by proliferation. Macrophage depletion and repopulation are thus decisive events characterizing the carcinogenic activity of particles and fibres.

Background Li-ion batteries (LIB) are increasingly used worldwide. They are made of low solubility micrometric particles, implying a potential for inhalation toxicity in occupational settings and possibly for consumers. LiCoO2 (LCO), one of the most used cathode material, induces inflammatory and fibrotic lung responses in mice. LCO also stabilizes hypoxia-inducible factor (HIF) -1α, a factor implicated in inflammation, fibrosis and carcinogenicity. Here, we investigated the role of cobalt, nickel and HIF-1α as determinants of toxicity, and evaluated their predictive value for the lung toxicity of LIB particles in in vitro assays. Results By testing a set of 5 selected LIB particles (LCO, LiNiMnCoO2, LiNiCoAlO2) with different cobalt and nickel contents, we found a positive correlation between their in vivo lung inflammatory activity, and (i) Co and Ni particle content and their bioaccessibility and (ii) the stabilization of HIF-1α in the lung. Inhibition of HIF-1α with chetomin or PX-478 blunted the lung inflammatory response to LCO in mice. In IL-1β deficient mice, HIF-1α was the upstream signal of the inflammatory lung response to LCO. In vitro, the level of HIF-1α stabilization induced by LIB particles in BEAS-2B cells correlated with the intensity of lung inflammation induced by the same particles in vivo. Conclusions We conclude that HIF-1α, stabilized in lung cells by released Co and Ni ions, is a mechanism-based biomarker of lung inflammatory responses induced by LIB particles containing Co/Ni. Documenting the Co/Ni content of LIB particles, their bioaccessibility and their capacity to stabilize HIF-1α in vitro can be used to predict the lung inflammatory potential of LIB particles.

Background: Silica continues to represent an intriguing topic of fundamental and applied research across various scientific fields, from geology to physics, chemistry, cell biology, and particle toxicology. The pathogenic activity of silica is variable, depending on the physico-chemical features of the particles. In the last 50 years, crystallinity and capacity to generate free radicals have been recognized as relevant features for silica toxicity. The ‘surface’ also plays an important role in silica toxicity, but this term has often been used in a very general way, without defining which properties of the surface are actually driving toxicity. How the chemical features (e.g., silanols and siloxanes) and configuration of the silica surface can trigger toxic responses remains incompletely understood. Main body: Recent developments in surface chemistry, cell biology and toxicology provide new avenues to improve our understanding of the molecular mechanisms of the adverse responses to silica particles. New physicochemical methods can finely characterize and quantify silanols at the surface of silica particles. Advanced computational modelling and atomic force microscopy offer unique opportunities to explore the intimate interactions between silica surface and membrane models or cells. In recent years, interdisciplinary research, using these tools, has built increasing evidence that surface silanols are critical determinants of the interaction between silica particles and biomolecules, membranes, cell systems, or animal models. It also has become clear that silanol configuration, and eventually biological responses, can be affected by impurities within the crystal structure, or coatings covering the particle surface. The discovery of new molecular targets of crystalline as well as amorphous silica particles in the immune system and in epithelial lung cells represents new possible toxicity pathways. Cellular recognition systems that detect specific features of the surface of silica particles have been identified. Conclusions: Interdisciplinary research bridging surface chemistry to toxicology is progressively solving the puzzling issue of the variable toxicity of silica. Further interdisciplinary research is ongoing to elucidate the intimate mechanisms of silica pathogenicity, to possibly mitigate or reduce surface reactivity. Keywords: Silica, Silicosis, Lung cancer, Auto-immune diseases, Surface reactivity, Silanol, Coating, Modelling, Spectroscopy, Atomic force microscopy

Resume Depuis deux decennies les nanotechnologies sont l’objet de toutes les attentions, avec des promesses revolutionnaires notamment en medecine. Dans l’agro-alimentaire, plusieurs applications connaissent un formidable essor, allant de produits phytosanitaires a des ingredients incorpores aux aliments ou dans les emballages alimentaires. Des nanoparticules minerales ou organiques (dimension comprise entre 1 et 100 nanometres) apportent des proprietes decuplees par rapport aux materiaux conventionnels. Cependant, qu’elles soient manufacturees et intentionnellement ajoutees le long de la chaine alimentaire ou generees lors des procedes de transformation des produits, les nanoparticules sont soupconnees de representer un risque pour la sante humaine. L’effet de taille permet a ces agents de passer les barrieres biologiques et de diffuser dans l’organisme, jusqu’a s’accumuler dans les organes systemiques ou leur forte reactivite chimique peut etre source de toxicite. Face aux applications nouvelles en alimentation, l’exposition orale a ces produits represente donc un nouveau defi en toxicologie.

CONTEXT: The addition of silver (Ag) to food items, and its migration from food packaging and appliances results in a dietary exposure in humans, estimated to 70-90 µg Ag/day. In view of the well-known bactericidal activity of Ag ions, concerns arise about a possible impact of dietary Ag on the gut microbiota (GM), which is a master determinant of human health and diseases. Repeated oral administration of Ag acetate (AgAc) can also cause systemic toxicity in rats with reported NOAELs of 4 mg AgAc/b.w./d for impaired fertility and 0.4 mg AgAc/b.w./d for developmental toxicity. OBJECTIVE: The objective of this study was to investigate whether oral exposure to AgAc can induce GM alterations at doses causing reproductive toxicity in rats. METHODS: Male and female Wistar rats were exposed during 10 weeks to AgAc incorporated into food (0, 0.4, 4 or 40 mg/kg b.w./d), and we analyzed the composition of the GM (α- and β-diversity). We documented bacterial function by measuring short-chain fatty acid (SCFA) production in cecal content. Ferroxidase activity, a biomarker of systemic Ag toxicity, was measured in serum. RESULTS AND CONCLUSIONS: From 4 mg/kg b.w./d onwards, we recorded systemic toxicity, as indicated by the reduction of serum ferroxidase activity, as well as serum Cu and Se concentrations. This systemic toxic response to AgAc might contribute to explain reprotoxic manifestations. We observed a dose-dependent modification of the GM composition in male rats exposed to AgAc. No impact of AgAc exposure on the production of bacterial SCFA was recorded. The limited GM changes recorded in this study do not appear related to a reprotoxicity outcome.

Background Ambient air pollution by particulate matters, including diesel exhaust particles (DEP), is a major cause of cardiovascular and metabolic mortality worldwide. The mechanisms by which DEP cause these adverse outcomes are not completely understood. Because the gut microbiota controls cardiovascular and metabolic health, we hypothesized that the fraction of inhaled DEP which reach the gut after mucociliary clearance and swallowing might induce gut dysbiosis and, in turn, contribute to aggravate or induce cardiovascular and metabolic diseases. Results Female ApoE−/− mice fed a Western diet, and wild-type (C57Bl/6) mice fed standard diet were gavaged with DEP (SRM2975) doses corresponding to mucociliary clearance from inhalation exposure (200 or 1000 ng/day, 3 times a week for 3 months; and 40, 200 or 1000 ng/day, 3 times a week for 6 months, respectively). No mortality, overt systemic or digestive toxicity was observed. A dose-dependent alteration of the gut microbiota was recorded in both strains. In ApoE−/−, β-diversity was modified by DEP, but no significant modification of the relative abundance of the phyla, families or genera was identified. In C57BL/6 mice, DEP reduced α-diversity (Shannon and Simpson indices), and modified β-diversity, including a reduction of the Proteobacteria and Patescibacteria phyla, and an increase of the Campylobacterota phylum. In both mouse models, perturbation of the gut microbiota composition was associated with a dose-dependent reduction of bacterial short chain fatty acids (butyrate and propionate) in cecal content. However, DEP ingestion did not aggravate (ApoE−/−), or induce (C57BL/6 mice) atherosclerotic plaques, and no metabolic alteration (glucose tolerance, resistance to insulin, or lipidemia) was recorded. Conclusions We show here that oral exposure to DEP, at doses relevant for human health, changes the composition and function of the gut microbiota. These modifications were, however, not translated into ultimate atherosclerotic or metabolic outcomes.

Background In vitro models are widely used in nanotoxicology. In these assays, a careful documentation of the fraction of nanomaterials that reaches the cells, i.e. the in vitro delivered dose, is a critical element for the interpretation of the data. The in vitro delivered dose can be measured by quantifying the amount of material in contact with the cells, or can be estimated by applying particokinetic models. For carbon nanotubes (CNTs), the determination of the in vitro delivered dose is not evident because their quantification in biological matrices is difficult, and particokinetic models are not adapted to high aspect ratio materials. Here, we applied a rapid and direct approach, based on femtosecond pulsed laser microscopy (FPLM), to assess the in vitro delivered dose of multi-walled CNTs (MWCNTs). Methods and results We incubated mouse lung fibroblasts (MLg) and differentiated human monocytic cells (THP-1) in 96-well plates for 24 h with a set of different MWCNTs. The cytotoxic response to the MWCNTs was evaluated using the WST-1 assay in both cell lines, and the pro-inflammatory response was determined by measuring the release of IL-1 beta by THP-1 cells. Contrasting cell responses were observed across the MWCNTs. The sedimentation rate of the different MWCNTs was assessed by monitoring turbidity decay with time in cell culture medium. These turbidity measurements revealed some differences among the MWCNT samples which, however, did not parallel the contrasting cell responses. FPLM measurements in cell culture wells revealed that the in vitro delivered MWCNT dose did not parallel sedimentation data, and suggested that cultured cells contributed to set up the delivered dose. The FPLM data allowed, for each MWCNT sample, an adjustment of the measured cytotoxicity and IL-1 beta responses to the delivered doses. This adjusted in vitro activity led to another toxicity ranking of the MWCNT samples as compared to the unadjusted activities. In macrophages, this adjusted ranking was consistent with existing knowledge on the impact of surface MWCNT functionalization on cytotoxicity, and might better reflect the intrinsic activity of the MWCNT samples. Conclusion The present study further highlights the need to estimate the in vitro delivered dose in cell culture experiments with nanomaterials. The FPLM measurement of the in vitro delivered dose of MWCNTs can enrich experimental results, and may refine our understanding of their interactions with cells. This work was supported by the Fonds de la Recherche scientifique - FNRS [Convention de recherche ERA-NET - No R.50.17.16.F], the Flemish Scientific Research Foundation [FWO; HB: 12P6819N], the U.S.A. National Science Foundation [grant CBET-1530505], and the German BMBF (FKZ: 03XP0063). Lison, D (corresponding author), Catholic Univ Louvain, Louvain Ctr Toxicol & Appl Pharmacol LTAP, Inst Rech Expt & Clin IREC, Brussels, Belgium. dominique.lison@uclouvain.be

Objectives Trace elements (TEs) from natural and anthropogenic sources are ubiquitous. Essential or not, their relevance for human health and disease is constantly expanding. Biological monitoring is a widely integrated tool in risk assessment both in occupational and environmental settings. However, the determination of appropriate and accurate reference values in the (specific) population is a prerequisite for a correct interpretation of biomonitoring data. This study aimed at determining the reference distribution for TEs (Al, As, Sb, Be, Bi, Cd, Co, Cu, Mn, Hg, Mo, Ni, Pb, Se, Tl, Sn, V, Zn) in the blood and/or plasma of the adult population in Belgium. Methods Blood and plasma samples were analyzed for 178 males and 202 females, recruited according to an a priori selection procedure, by inductively coupled plasma mass spectrometry (ICP-MS). Results Reference values were established with high confidence for AsT, Cd, Cu, HgT, Mn, Mo, Pb, Sn, Se, Tl and Zn. Compared to previously published data in the Belgian population, a decreasing time trend is observed for Zn, Cd and Pb. Globally, the results also indicate that the current exposure levels to TEs in the Belgian population are similar to those from other recent national surveys. Conclusions These reference values and limits obtained through validated analytical and statistical methods will be useful for future occupational and/or environmental surveys. They will contribute to decision-making concerning both public health policies but also exposure assessments on an individual scale.

Background The terms agglomerates and aggregates are frequently used in the regulatory definition(s) of nanomaterials (NMs) and hence attract attention in view of their potential influence on health effects. However, the influence of nanoparticle (NP) agglomeration and aggregation on toxicity is poorly understood although it is strongly believed that smaller the size of the NPs greater the toxicity. A toxicologically relevant definition of NMs is therefore not yet available, which affects not only the risk assessment process but also hinders the regulation of nano-products. In this study, we assessed the influence of NP agglomeration on their toxicity/biological responses in vitro and in vivo. Results We tested two TiO2 NPs with different primary sizes (17 and 117 nm) and prepared ad-hoc suspensions composed of small or large agglomerates with similar dispersion medium composition. For in vitro testing, human bronchial epithelial (HBE), colon epithelial (Caco2) and monocytic (THP-1) cell lines were exposed to these suspensions for 24 h and endpoints such as cytotoxicity, total glutathione, epithelial barrier integrity, inflammatory mediators and DNA damage were measured. Large agglomerates of 17 nm TiO2 induced stronger responses than small agglomerates for glutathione depletion, IL-8 and IL-1β increase, and DNA damage in THP-1, while no effect of agglomeration was observed with 117 nm TiO2. In vivo, C57BL/6JRj mice were exposed via oropharyngeal aspiration or oral gavage to TiO2 suspensions and, after 3 days, biological parameters including cytotoxicity, inflammatory cell recruitment, DNA damage and biopersistence were measured. Mainly, we observed that large agglomerates of 117 nm TiO2 induced higher pulmonary responses in aspirated mice and blood DNA damage in gavaged mice compared to small agglomerates. Conclusion Agglomeration of TiO2 NPs influences their toxicity/biological responses and, large agglomerates do not appear less active than small agglomerates. This study provides a deeper insight on the toxicological relevance of NP agglomerates and contributes to the establishment of a toxicologically relevant definition for NMs.

Background Li-ion batteries (LIB) are used in most portable electronics. Among a wide variety of materials, LiCoO2 (LCO) is one of the most used for the cathode of LIB. LCO particles induce oxidative stress in mouse lungs due to their Co content, and have a strong inflammatory potential. In this study, we assessed the mutagenic potential of LCO particles in lung cells in comparison to another particulate material used in LIB, LTO (Li4Ti5O12), which has a low inflammatory potential compared to LCO particles. Results We assessed the mutagenic potential of LCO and LTO particles in vitro by performing a cytokinesis-block micronucleus (MN) assay with rat lung epithelial cells (RLE), as well as in vivo in alveolar type II epithelial (AT-II) cells. LCO particles induced MN in vitro at non-cytotoxic concentrations and in vivo at non-inflammatory doses, indicating a primary genotoxic mechanism. LTO particles did not induce MN. Electron paramagnetic resonance and terephthalate assays showed that LCO particles produce hydroxyl radicals (•OH). Catalase inhibits this •OH production. In an alkaline comet assay with the oxidative DNA damage repair enzyme human 8-oxoguanine DNA glycosylase 1, LCO particles induced DNA strand breaks and oxidative lesions. The addition of catalase reduced the frequency of MN induced by LCO particles in vitro. Conclusions We report the mutagenic activity of LCO particles used in LIB in vitro and in vivo. Our data support the role of Co(II) ions released from these particles in their primary genotoxic activity which includes the formation of •OH by a Fenton-like reaction, oxidative DNA lesions and strand breaks, thus leading to chromosomal breaks and the formation of MN. Documenting the genotoxic potential of the other LIB particles, especially those containing Co and/or Ni, is therefore needed to guarantee a safe and sustainable development of LIB.

Significance Silica particles with a population of nearly free silanols damage cellular membranes and initiate inflammatory reactions. Nearly free silanols are found on the surface of both fractured quartz and amorphous silica particles, and their occurrence initiates the toxicity of silica, thus revisiting the ancient paradigm whereby crystallinity is critical for silica toxicity. This finding resolves the lingering questions about the origin and the variability of the toxicity of silica particles. The discovery of the biological activity of nearly free silanols opens perspectives for the prevention of silicosis through a safer-by-design approach, and will have an impact on other fields that involve interfacial phenomena, including biomaterial design, nanofabrication, and catalysis., Inhalation of silica particles can induce inflammatory lung reactions that lead to silicosis and/or lung cancer when the particles are biopersistent. This toxic activity of silica dusts is extremely variable depending on their source and preparation methods. The exact molecular moiety that explains and predicts this variable toxicity of silica remains elusive. Here, we have identified a unique subfamily of silanols as the major determinant of silica particle toxicity. This population of “nearly free silanols” (NFS) appears on the surface of quartz particles upon fracture and can be modulated by thermal treatments. Density functional theory calculations indicates that NFS locate at an intersilanol distance of 4.00 to 6.00 Å and form weak mutual interactions. Thus, NFS could act as an energetically favorable moiety at the surface of silica for establishing interactions with cell membrane components to initiate toxicity. With ad hoc prepared model quartz particles enriched or depleted in NFS, we demonstrate that NFS drive toxicity, including membranolysis, in vitro proinflammatory activity, and lung inflammation. The toxic activity of NFS is confirmed with pyrogenic and vitreous amorphous silica particles, and industrial quartz samples with noncontrolled surfaces. Our results identify the missing key molecular moieties of the silica surface that initiate interactions with cell membranes, leading to pathological outcomes. NFS may explain other important interfacial processes involving silica particles.

Cymbopogon giganteus Chiov. (Poaceae) is a medicinal plant used to treat various diseases in traditional medicine in several African countries. The present study aims to evaluate the oral and inhalation toxicity as well as the mutagenic effects of the essential oil of Cymbopogon giganteus leaves (EOCG) from a sample collected in Benin. Mutagenic potential was assessed by the Ames test using Salmonella typhimurium strains TA98 and TA100. Oral acute toxicity was carried out by administration of a single dose of 2000 mg/kg b.w. to Wistar rats while oral subacute toxicity was assessed by daily administration of 50 and 500 mg/kg of EOCG for 28 days. Finally, inhalation toxicity was assessed by administration of a single dose of 0.125%, 0.5%, 2% or 5% v/v of EOCG emulsions in 0.05% v/v lecithin solution in sterile water for the first experiment, and in a second one by administration of single dose of 0.125% or 0.5% v/v. A broncho-alveolar lavage was performed after 3 h or 24 h, respectively. The results show that EOCG is not mutagenic on Salmonella typhimurium strains at the highest concentration tested (200 μg/plate). In the acute oral toxicity study, EOCG induce neither mortality nor toxicity, showing that the LD50 is greater than 2000 mg/kg. The subacute oral toxicity study at both doses did not show any significant difference in body weight, relative organ weight, hematological and/or biochemical parameters or histopathology as compared to the control group. EOCG induced mortality and inflammation in lungs 3 h after administration of a single dose of 5% or 2% v/v. Single doses of 0.125% or 0.5% v/v did not induce inflammation, cell recruitment nor cytotoxicity in lungs 3 h or 24 h after administration, suggesting safety at these concentrations. This first report on the in vivo toxicity will be useful to guide safe uses of EOCG.

Fe-based materials are currently considered for manufacturing biodegradable coronary stents. Here we show that Fe has a strong potential to generate hydroxyl radicals (HO·) during corrosion. This HO· generation, but not corrosion, can be inhibited by catalase. Oxidative stress was observed (increased HO-1 expression) in aortic rings after direct exposure to Fe, but not in the presence of catalase or after indirect exposure. This oxidative stress response induced an uncoupling of eNOS in, and a consequent reduced NO production by endothelial cells exposed to Fe. In isolated rat aortic rings NO production was also reduced by HO· generated during Fe corrosion, as indicated by the protective role of catalase. Finally, all these mechanisms contributed to impaired endothelium- dependent relaxation in aortic rings caused by HO· generated during the direct contact with Fe. This deleterious impact of Fe corrosion on the endothelial function should be integrated when considering the use of biodegradable Fe-based alloys for vascular implants.

Heavy metal screening is currently very popular, especially on the web, and has become a “must do” for an increasing number of patients. The relative availability of inductively coupled plasma mass spectrometry equipments capable of measuring multiple elements at once has boosted the diffusion of these practices. In most cases, the list of elements measured in these screens reflects the analytical capacity of the lab more than sound toxicological considerations. These “urine mobilization tests”, “post-chelator challenges”, “provocative chelations”, “chelation challenges”, or “challenge tests” (hereafter called “provocative chelation test” [PCT]) represent an appreciable business. The screen almost systematically reveals “heavy metal poisoning”, and “detox cures” are generally recommended.[...]

Fe-based materials have increasingly been considered for the development of biodegradable cardiovascular stents. A wide range of in vitro and in vivo studies should be done to fully evaluate their biocompatibility. In this review, we summarized and analyzed the findings and the methodologies used to assess the biocompatibility of Fe materials. The majority of investigators drew conclusions about in vitro Fe toxicity based on indirect contact results. The setup applied in these tests seems to overlook the possible effects of Fe corrosion and does not allow for understanding of the complexity of released chemical forms and their possible impact on tissue. It is in particular important to ensure that test setups or interpretations of in vitro results do not hide some important mechanisms, leading to inappropriate subsequent in vivo experiments. On the other hand, the sample size of existing in vivo implantations is often limited, and effects such as local toxicity or endothelial function are not deeply scrutinized. The main advantages and limitations of in vitro design strategies applied in the development of Fe-based alloys and the correlation with in vivo studies are discussed. It is evident from this literature review that we are not yet ready to define an Fe-based material as safe or biocompatible.

This article summarizes recent experimental and epidemiological data on the genotoxic and carcinogenic activities of cobalt compounds. Emphasis is on the respiratory system, but endogenous exposure from Co-containing alloys used in endoprostheses, and limited data on nanomaterials and oral exposures are also considered. Two groups of cobalt compounds are differentiated on the basis of their mechanisms of toxicity: (1) those essentially involving the solubilization of Co(II) ions, and (2) metallic materials for which both surface corrosion and release of Co(II) ions act in concert. For both groups, identified genotoxic and carcinogenic mechanisms are non-stochastic and thus expected to exhibit a threshold. Cobalt compounds should, therefore, be considered as genotoxic carcinogens with a practical threshold. Accumulating evidence indicates that chronic inhalation of cobalt compounds can induce respiratory tumors locally. No evidence of systemic carcinogenicity upon inhalation, oral or endogenous exposure is available. The scarce data available for Co-based nanosized materials does not allow deriving a specific mode of action or assessment for these species.

Background Nanotechnologies provide new opportunities for improving the safety, quality, shelf life, flavor and appearance of foods. The most common nanoparticles (NPs) in human diet are silver metal, mainly present in food packaging and appliances, and silicon and titanium dioxides used as additives. The rapid development and commercialization of consumer products containing these engineered NPs is, however, not well supported by appropriate toxicological studies and risk assessment. Local and systemic toxicity and/or disruption of the gut microbiota (GM) have already been observed after oral administration of NPs in experimental animals, but results are not consistent and doses used were often much higher than the estimated human intakes. In view of the strong evidence linking alterations of the GM to cardiometabolic (CM) diseases, we hypothesized that dietary NPs might disturb this GM-CM axis. Materials and methods We exposed male C57BL/6JRj mice (n = 13 per dose group) to dietary NPs mixed in food pellets at doses relevant for human exposure: Ag (0, 4, 40 or 400 μg/kg pellet), SiO2 (0, 0.8, 8 and 80 mg/kg pellet) or TiO2 (0, 0.4, 4 or 40 mg/kg pellet). After 24 weeks of exposure, we assessed effects on the GM and CM health (n = 8 per dose group). The reversibility of the effects was examined after 8 additional weeks without NPs exposure (recovery period, n ≤ -5 per dose group). Results No overt toxicity was recorded. The GM β-diversity was dose-dependently disrupted by the three NPs, and the bacterial short chain fatty acids (SCFAs) were dose-dependently reduced after the administration of SiO2 and TiO2 NPs. These effects disappeared completely or partly after the recovery period, strengthening the association with dietary NPs. We did not observe atheromatous disease or glucose intolerance after NP exposure. Instead, dose-dependent decreases in the expression of IL-6 in the liver, circulating triglycerides (TG) and urea nitrogen (BUN) were recorded after administration of the NPs. Conclusion We found that long-term oral exposure to dietary NPs at doses relevant for estimated human intakes disrupts the GM composition and function. These modifications did not appear associated with atheromatous or deleterious metabolic outcomes.

Monocytes infiltrating scar tissue are predominantly viewed as progenitor cells. Here, we show that tissue CCR2+ monocytes have specific immunosuppressive and profibrotic functions. CCR2+ monocytic cells are acutely recruited to the lung before the onset of silica-induced fibrosis in mice. These tissue monocytes are defined as monocytic myeloid-derived suppressor cells (M-MDSCs) because they significantly suppress T-lymphocyte proliferation in vitro. M-MDSCs collected from silica-treated mice also express transforming growth factor (TGF)-β1, which stimulates lung fibroblasts to release tissue inhibitor of metalloproteinase (TIMP)-1, an inhibitor of metalloproteinase collagenolytic activity. By using LysMCreCCR2loxP/loxP mice, we show that limiting CCR2+ M-MDSC accumulation reduces the pulmonary contents of TGF-β1, TIMP-1 and collagen after silica treatment. M-MDSCs do not differentiate into lung macrophages, granulocytes or fibrocytes during pulmonary fibrogenesis. Collectively, our data indicate that M-MDSCs contribute to lung fibrosis by specifically promoting a non-degrading collagen microenvironment. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Among the physico-chemical features responsible for the so-called “variability of quartz hazard”, a key role has been assigned to the silica surface charge, evaluated by means of ζ potential measurement. The ζ potential of silica describes the protonation state of silanols which, in turn, determine interactions with cell membranes. To gain a molecular understanding of the role of silanols in silica pathogenicity, we conducted a systematic investigation of the variation of the ζ potential as a function of pH (ζ plot titration curve) on a large set of respirable quartz particles with different levels of metal contaminants. The membranolytic activity of the particles on red blood cells, used as a readout of pathogenic activity, was assessed in parallel. Pure quartz surfaces showed sigmoid-shaped ζ plots suggesting the presence of silanol families with similar acidity, whereas contaminated dusts exhibited convex-shaped ζ plots, indicating a higher silanol heterogeneity on contaminated surfaces with respect to the pure ones. The quartz particles with a higher surface heterogeneity related to metal contamination showed a higher membranolytic activity. By removing structural defects and chemical heterogeneity, the ζ plot shifted towards the typical shape of pure quartz and the membranolytic activity was reduced. We conclude that the ζ plot is a useful readout to measure the acid-base behavior of quartz surfaces and to describe the chemical heterogeneity of quartz silanols. Surface heterogeneity, here induced by metal contamination, is proposed as the main cause of quartz membranolytic activity, further supporting the hypothesis that surface silanol disorganization determines silica pathogenicity.

Inhalation of respirable crystalline silica (RCS) can lead to serious health effects such as silicosis and lung cancer. There also seems to be a general consensus to consider that RCS exposure is associated with obstructive lung impairment or chronic obstructive pulmonary disease (COPD), a leading cause of mortality, morbidity, and disability worldwide. It is, however, not clear whether occupational exposure levels (OELs), generally set to prevent silicosis, also protect workers from developing an obstructive impairment. This review aims at clarifying the potential relationship between RCS exposure and obstructive lung impairment as defined by spirometry. Eleven studies that reported both silica exposure levels and spirometry results were identified. We systematically extracted data pertaining to (a) the population studied, (b) level of exposure to RCS and other pollutants, (c) spirometry procedure and interpretation, and (d) methodology used to investigate the relationship between RCS exposure and spirometry. These studies add supporting evidence in favor of a qualitative association between occupational activities exposing to RCS and obstructive lung dysfunction. However, no well-founded quantitative estimate can be drawn from these investigations; the available relevant literature does not allow defining a RCS exposure threshold associated with an increased risk of obstructive lung dysfunction, as defined by spirometry, in workers without silicosis. Further research is needed, but, as highlighted in this review, conducting epidemiological studies with both valid exposure and outcome measurements is a real challenge.

The WST-1 assay is the most common test to assess the in vitro cytotoxicity of chemicals. Tetrazolium-based assays can, however, be affected by the interference of tested chemicals, including carbon nanotubes or Mg particles. Here, we report a new interference of Mn materials with the WST-1 assay. Endothelial cells exposed to Mn particles (Mn alone or Fe-Mn alloy from 50 to 1600 μg/ml) were severely damaged according to the WST-1 assay, but not the ATP content assay. Subsequent experiments revealed that Mn particles interfere with the reduction of the tetrazolium salt to formazan. Therefore, the WST-1 assay is not suitable to evaluate the in vitro cytotoxicity of Mn-containing materials, and luminescence-based assays such as CellTiter-Glo® appear more appropriate.

Background Li-ion batteries (LIB) are used in most portable electronics. Among a wide variety of materials, LiCoO2 (LCO) is one of the most used for the cathode of LIB. LCO particles induce oxidative stress in mouse lungs due to their Co content, and have a strong inflammatory potential. In this study, we assessed the mutagenic potential of LCO particles in lung cells in comparison to another particulate material used in LIB, LTO (Li4Ti5O12), which has a low inflammatory potential compared to LCO particles. Results We assessed the mutagenic potential of LCO and LTO particles in vitro by performing a cytokinesis-block micronucleus (MN) assay with rat lung epithelial cells (RLE), as well as in vivo in alveolar type II epithelial (AT-II) cells. LCO particles induced MN in vitro at non-cytotoxic concentrations and in vivo at non-inflammatory doses, indicating a primary genotoxic mechanism. LTO particles did not induce MN. Electron paramagnetic resonance and terephthalate assays showed that LCO particles produce hydroxyl radicals (•OH). Catalase inhibits this •OH production. In an alkaline comet assay with the oxidative DNA damage repair enzyme human 8-oxoguanine DNA glycosylase 1, LCO particles induced DNA strand breaks and oxidative lesions. The addition of catalase reduced the frequency of MN induced by LCO particles in vitro. Conclusions We report the mutagenic activity of LCO particles used in LIB in vitro and in vivo. Our data support the role of Co(II) ions released from these particles in their primary genotoxic activity which includes the formation of •OH by a Fenton-like reaction, oxidative DNA lesions and strand breaks, thus leading to chromosomal breaks and the formation of MN. Documenting the genotoxic potential of the other LIB particles, especially those containing Co and/or Ni, is therefore needed to guarantee a safe and sustainable development of LIB.

International audience; Background: The role that pesticides in the domestic environment might play in the etiology of childhood leukemia remains a subject of controversy. Recent studies often reached inconsistent conclusions. Objective: To update our earlier systematic review on the association between residential/household/domestic exposure to pesticides and childhood leukemia, and to explore potential sources of heterogeneity not previously assessed. Methods: A systematic search of studies published in English between January 2009 and June 2018 was conducted in MEDLINE, and a “snowball searching” was performed from the reference list of identified publications and from Web of Science citations. Risk estimates were extracted from 15 case-control studies published between 1987 and 2018. The quality of the publications was assessed by using a modified version of the Downs and Black (1998) checklist. A random-effect meta-analysis model was used to calculate summary odds ratios (SOR) and separate analyses were conducted for acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), unspecified AL/leukemia and any leukemia types. Stratification by critical exposure period, exposure location, pesticide biocide category, child age at diagnosis, study quality, specific exposures, type of pest treated, and geographic location were performed. Results: A statistically significant association between residential pesticide exposure and childhood leukemia was observed by combining all studies (SOR: 1.57; 95% CI: 1.27–1.95) without evidence of publication bias. Statistically significant increased risks were observed for all types of leukemia, and specifically for exposure during pregnancy, indoor exposure, prenatal exposure to insecticides and whatever the age at diagnosis. Statistical significance was also reached for high quality studies, pet treatments, professional pest control treatment and use of insect repellants, mosquito treatment and for studies from USA/Canada or International. The highest increased risks were observed for AML among children aged 2 years or less, as well as for unspecified leukemia type observed after prenatal indoor exposure. Conclusions: A positive association between domestic pesticide exposure and childhood leukemia is confirmed. Although the literature provides moderate to low-quality of evidence, these new results further justify the need of limiting the use of household pesticides during pregnancy and childhood.

Background: With tobacco smoking, diet is the main source of cadmium (Cd) exposure in the general population. The carcinogenic and estrogenic activities of Cd make it a contaminant of potential concern for hormone-dependent cancers including breast cancer. Postmenopausal women represent the most appropriate population to investigate the possible impact of exogenous factors with potential estrogenic activity on breast cancer as, after menopause, their estrogenic influence is predominant. Objectives: We systematically reviewed available studies on the association between dietary exposure to Cd and breast cancer focusing on postmenopausal women. A meta-analysis combining the risk estimators was performed and potential sources of between studies heterogeneity were traced. Methods: Studies were searched from MEDLINE through 31 January 2015 and from the reference lists of relevant publications. Six eligible studies published between 2012 and 2014 were identified and relative risk estimates were extracted. Meta-rate ratio estimates (mRR) were calculated according to fixed and random-effect models. Meta-analyses were performed on the whole set of data and separate analyses were conducted after stratification for study design, geographic location, use of hormone replacement therapy (HRT), tumor estrogen receptor status (ER+ or ER−), progesterone receptor status (PGR+ or PGR−), body mass index (BMI), smoker status, zinc or iron intake. Results: No statistically significant increased risk of breast cancer was observed when all studies were combined (mRR = 1.03; 95% confidence interval [CI]: 0.89–1.19). Several sources of heterogeneity and inconsistency were identified, including smoker status, HRT use, BMI, zinc and iron intake. Inconsistency was also strongly reduced when only considering ER−, PGR−, tumors subgroups from USA and from Japan. The risks were, however, not substantially modified after stratifications. No evidence of publication bias was found. Conclusion: The present study does not provide support for the hypothesis that dietary exposure to Cd increases the risk of breast cancer in postmenopausal women. Misclassification in dietary Cd assessment in primary studies could have biased the results towards a finding of no association. Keywords: Dietary cadmium, Breast cancer, Postmenopausal women, Systematic review, Meta-analysis

Fe-based materials are considered for the manufacture of temporary implants that degrade through the corrosion of Fe by oxygen. Here we document the generation of hydroxyl radicals (HO˙) during this corrosion process, and their deleterious impacts on human endothelial (ECs) and smooth muscle cells (SMCs) in vitro. The generation of HO˙ was documented by two independent acellular assays, terephtalic acid hydroxylation (fluorescence) and spin trapping technique coupled with electron paramagnetic resonance spectroscopy. All Fe-based materials tested exhibited a strong potential to generate HO˙. The addition of catalase prevented the formation of HO˙. Cellular responses were assessed in two ECs and SMCs lines using different cytotoxicity assays (WST-1 and CellTiter-Glo). Cells were exposed directly to Fe powder in the presence/absence of catalase, or to extracts obtained from the corrosion of Fe. Cell viability was dose-dependently affected by the direct contact with Fe materials, but not in the presence of catalase or after indirect exposure to cell extracts. The deleterious effect of HO˙ on ECs and SMCs was confirmed by the dose-dependent increase of the transcripts of the oxidative stress gene heme oxygenase-1 4 h or 6 h after direct exposure to the particles, but not in presence of catalase or after indirect exposure. The demonstration of HO˙production during corrosion and consequent oxidative stress on human ECs and SMCs newly reveals a deleterious consequence of Fe-corrosion that should be integrated in the assessment of the biocompatibility of Fe-based alloys.