Inflammation as a Key Outcome Pathway in Particle Induced Effects in the Lung.

Inflammation is considered a key event in the pathology of many chronic diseases, including pulmonary and systemic particle induced effects. In addition, inflammation is now considered as the key response in standard setting for poorly-soluble low toxicity (PSLT) particles and also the critical endpoint to screen for in OECD based sub-chronic animal inhalation testing protocols. During Particles & Health 2021, an afternoon session was dedicated to the subject and a brief summary of the most important messages are summarized in this paper. In the first part of this session, two speakers (Prof. Lison and Dr Duffin) provided state of the art insight into different aspects and sequels to (persistent) inflammation as a protective or adverse response. Most recent insights on the role of different macrophage cell types were presented as well as perspectives and data provided by inflammatory pathways in humans, such as in asthma and COPD. A brief review of the expert workshop on PSLT particles focusing on the regulatory impact of using persistent inflammation as a key outcome was provided by Kevin Driscoll. The second part of the session focused on the outcomes that are associated with inflammation in animal studies, with an emphasis by Drs. Harkema (Michigan State) and Weber (Anapath) on cell proliferation and other pathologies that need to be considered when comparing human and animal responses, such as outcomes from 14- or 28 day inhalation studies used for specific target organ toxicity classification.

Monocytic Ontogeny of Regenerated Macrophages Characterizes the Mesotheliomagenic Responses to Carbon Nanotubes.

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.

Dietary nanoparticles alter the composition and function of the gut microbiota in mice at dose levels relevant for human exposure.

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 ß-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.

Femtosecond pulsed laser microscopy: a new tool to assess the in vitro delivered dose of carbon nanotubes in cell culture experiments.

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-1ß 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-1ß 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.

The pulmonary toxicity of carboxylated or aminated multi-walled carbon nanotubes in mice is determined by the prior purification method.

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-1ß, IL-6, CXCL1) in BALF, while thermal purification had a greater effect on pro-fibrotic cytokines (CCL2, OPN, TGF-ß1). 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.

Nearly free surface silanols are the critical molecular moieties that initiate the toxicity of silica particles.

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.

In Vitro and In Vivo Toxicity Studies on Cymbopogon giganteus Chiov. Leaves Essential Oil from Benin.

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.

Agglomeration of titanium dioxide nanoparticles increases toxicological responses in vitro and in vivo.

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-1ß 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.

Is aggregated synthetic amorphous silica toxicologically relevant?

BACKGROUND: The regulatory definition(s) of nanomaterials (NMs) frequently uses the term 'agglomerates and aggregates' (AA) despite the paucity of evidence that AA are significantly relevant from a nanotoxicological perspective. This knowledge gap greatly affects the safety assessment and regulation of NMs, such as synthetic amorphous silica (SAS). SAS is used in a large panel of industrial applications. They are primarily produced as nano-sized particles (1-100 nm in diameter) and considered safe as they form large aggregates (> 100 nm) during the production process. So far, it is indeed believed that large aggregates represent a weaker hazard compared to their nano counterpart. Thus, we assessed the impact of SAS aggregation on in vitro cytotoxicity/biological activity to address the toxicological relevance of aggregates of different sizes. RESULTS: We used a precipitated SAS dispersed by different methods, generating 4 ad-hoc suspensions with different aggregate size distributions. Their effect on cell metabolic activity, cell viability, epithelial barrier integrity, total glutathione content and, IL-8 and IL-6 secretion were investigated after 24 h exposure in human bronchial epithelial (HBE), colon epithelial (Caco2) and monocytic cells (THP-1). We observed that the de-aggregated suspension (DE-AGGR), predominantly composed of nano-sized aggregates, induced stronger effects in all the cell lines than the aggregated suspension (AGGR). We then compared DE-AGGR with 2 suspensions fractionated from AGGR: the precipitated fraction (PREC) and the supernatant fraction (SuperN). Very large aggregates in PREC were found to be the least cytotoxic/biologically active compared to other suspensions. SuperN, which contains aggregates larger in size (> 100 nm) than in DE-AGGR but smaller than PREC, exhibited similar activity as DE-AGGR. CONCLUSION: Overall, aggregation resulted in reduced toxicological activity of SAS. However, when comparing aggregates of different sizes, it appeared that aggregates > 100 nm were not necessarily less cytotoxic than their nano-sized counterparts. This study suggests that aggregates of SAS are toxicologically relevant for the definition of NMs.

HIF-1α is a key mediator of the lung inflammatory potential of lithium-ion battery particles.

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-1ß 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.

The puzzling issue of silica toxicity: are silanols bridging the gaps between surface states and pathogenicity?

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 physico-chemical 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.

Household exposure to pesticides and risk of leukemia in children and adolescents: Updated systematic review and meta-analysis.

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.

New interplay between interstitial and alveolar macrophages explains pulmonary alveolar proteinosis (PAP) induced by indium tin oxide particles.

Occupational exposure to indium tin oxide (ITO) particles has been associated with the development of severe lung diseases, including pulmonary alveolar proteinosis (PAP). The mechanisms of this lung toxicity remain unknown. Here, we reveal the respective roles of resident alveolar (Siglec-Fhigh AM) and recruited interstitial (Siglec-Flow IM) macrophages contributing in concert to the development of PAP. In mice treated with ITO particles, PAP is specifically associated with IL-1α (not GM-CSF) deficiency and Siglec-Fhigh AM (not Siglec-Flow IM) depletion. Mechanistically, ITO particles are preferentially phagocytosed and dissolved to soluble In3+ by Siglec-Flow IM. In contrast, Siglec-Fhigh AM weakly phagocytose or dissolve ITO particles, but are sensitive to released In3+ through the expression of the transferrin receptor-1 (TfR1). Blocking pulmonary Siglec-Flow IM recruitment in CCR2-deficient mice reduces ITO particle dissolution, In3+ release, Siglec-Fhigh AM depletion, and PAP formation. Restoration of IL-1-related Siglec-Fhigh AM also prevented ITO-induced PAP. We identified a new mechanism of secondary PAP development according to which metal ions released from inhaled particles by phagocytic IM disturb IL-1α-dependent AM self-maintenance and, in turn, alveolar clearance.

Residential exposure to pesticides as risk factor for childhood and young adult brain tumors: A systematic review and meta-analysis.

BACKGROUND: Accumulating evidence suggests a positive association between exposure to non-agricultural pesticides and childhood brain tumors (CBT). OBJECTIVE: (1) To conduct a systematic review and meta-analysis of published studies on the association between residential/household/domestic exposure to pesticides and childhood brain tumors. (2) To clarify variables that could impact the results. METHODS: Publications in English were identified from a MEDLINE search through 28 February 2017 and from the reference list of identified publications. Risk estimates were extracted from 18 case-control studies published between 1979 and 2016 and study quality assessments were performed. Summary odds ratios (mOR) were calculated according to fixed and random-effect meta-analysis models. Separate analyses were conducted after stratification for study quality, critical exposure period, exposure location, specific exposures, pesticide category, application methods, type of pest treated, type of CBT, child's age at diagnosis and geographic location. RESULTS: Statistically significant associations were observed with CBT after combining all studies (mOR: 1.26; 95% CI: 1.13-1.40) without evidence of inconsistency between study results or publication bias. Specifically, increased risks were observed for several groupings and more particularly for gliomas and exposure involving insecticides. Statistical significance was also reached for high quality studies, for all exposure periods, for indoor exposure and, more particularly, during the prenatal period for all stratifications involving insecticides (except for outdoor use), for pet treatments, for flea/tick treatment, for studies from USA/Canada and studies from Europe (borderline) as well as for data from studies including children of up to 10years at diagnosis and of up to 15years. CONCLUSIONS: Our findings support an association between residential exposure to pesticides and childhood brain tumors. Although causality cannot be established, these results add to the evidence leading to recommend limiting residential use of pesticides and to support public health policies serving this objective.

Do current OELs for silica protect from obstructive lung impairment? A critical review of epidemiological data.

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.

Mesothelioma response to carbon nanotubes is associated with an early and selective accumulation of immunosuppressive monocytic cells.

BACKGROUND: The asbestos-like toxicity of some engineered carbon nanotubes (CNT), notably their capacity to induce mesothelioma, is a serious cause of concern for public health. Here we show that carcinogenic CNT induce an early and sustained immunosuppressive response characterized by the accumulation of monocytic Myeloid Derived Suppressor Cells (M-MDSC) that counteract effective immune surveillance of tumor cells. METHODS: Wistar rats and C57BL/6 mice were intraperitoneally injected with carcinogenic multi-walled Mitsui-7 CNT (CNT-7) or crocidolite asbestos. Peritoneal mesothelioma development and immune cell accumulation were assessed until 12 months. Leukocyte sub-populations were identified by recording expression of CD11b/c and His48 by flow cytometry. The immunosuppressive activity on T lymphocytes of purified peritoneal leukocytes was assessed in a co-culture assay with activated spleen cells. RESULTS: We demonstrate that long and short mesotheliomagenic CNT-7 injected in the peritoneal cavity of rats induced, like asbestos, an early and selective accumulation of monocytic cells (CD11b/c(int) and His48(hi)) which possess the ability to suppress polyclonal activation of T lymphocytes and correspond to M-MDSC. Peritoneal M-MDSC persisted during the development of peritoneal mesothelioma in CNT-7-treated rats but were only transiently recruited after non-carcinogenic CNT (CNT-M, CNT-T) injection. Peritoneal M-MDSC did not accumulate in mice which are resistant to mesothelioma development. CONCLUSIONS: Our data provide new insights into the initial pathogenic events induced by CNT, adding a new component to the adverse outcome pathway leading to mesothelioma development. The specificity of the M-MDSC response after carcinogenic CNT exposure highlights the interest of this response for detecting the ability of new nanomaterials to cause cancer.

The complex cascade of cellular events governing inflammasome activation and IL-1ß processing in response to inhaled particles.

The innate immune system is the first line of defense against inhaled particles. Macrophages serve important roles in particle clearance and inflammatory reactions. Following recognition and internalization by phagocytes, particles are taken up in vesicular phagolysosomes. Intracellular phagosomal leakage, redox unbalance and ionic movements induced by toxic particles result in pro-IL-1ß expression, inflammasome complex engagement, caspase-1 activation, pro-IL-1ß cleavage, biologically-active IL-1ß release and finally inflammatory cell death termed pyroptosis. In this review, we summarize the emerging signals and pathways involved in the expression, maturation and secretion of IL-1ß during these responses to particles. We also highlight physicochemical characteristics of particles (size, surface and shape) which determine their capacity to induce inflammasome activation and IL-1ß processing.

Revisiting the paradigm of silica pathogenicity with synthetic quartz crystals: the role of crystallinity and surface disorder.

BACKGROUND: Exposure to some - but not all - quartz particles is associated to silicosis, lung cancer and autoimmune diseases. What imparts pathogenicity to any single quartz source is however still unclear. Crystallinity and various surface features are implied in toxicity. Quartz dusts used so far in particle toxicology have been obtained by grinding rocks containing natural quartz, a process which affects crystallinity and yields dusts with variable surface states. To clarify the role of crystallinity in quartz pathogenicity we have grown intact quartz crystals in respirable size. METHODS: Quartz crystals were grown and compared with a fractured specimen obtained by grinding the largest synthetic crystals and a mineral quartz (positive control). The key physico-chemical features relevant to particle toxicity - particle size distribution, micromorphology, crystallinity, surface charge, cell-free oxidative potential - were evaluated. Membranolysis was assessed on biological and artificial membranes. Endpoints of cellular stress were evaluated on RAW 264.7 murine macrophages by High Content Analysis after ascertaining cellular uptake by bio-TEM imaging of quartz-exposed cells. RESULTS: Quartz crystals were grown in the submicron (n-Qz-syn) or micron (µ-Qz-syn) range by modulating the synthetic procedure. Independently from size as-grown quartz crystals with regular intact faces did not elicit cellular toxicity and lysosomal stress on RAW 264.7 macrophages, and were non-membranolytic on liposome and red blood cells. When fractured, synthetic quartz (µ-Qz-syn-f) attained particle morphology and size close to the mineral quartz dust (Qz-f, positive control) and similarly induced cellular toxicity and membranolysis. Fracturing imparted a higher heterogeneity of silanol acidic sites and radical species at the quartz surface. CONCLUSIONS: Our data support the hypothesis that the biological activity of quartz dust is not due to crystallinity but to crystal fragmentation, when conchoidal fractures are formed. Besides radical generation, fracturing upsets the expected long-range order of non-radical surface moieties - silanols, silanolates, siloxanes - which disrupt membranes and induce cellular toxicity, both outcomes associated to the inflammatory response to quartz.