An integrated new approach methodology for inhalation risk assessment of safe and sustainable by design nanomaterials.

The production and use of nanomaterials (NMs) has increased over the last decades posing relevant questions on their risk after release and exposure of the population or sub-populations. In this context, the safe and sustainable by design (SSbD) approach framework requires to assess the potential hazard connected with intrinsic properties of the material along the whole life cycle of the NM and/or of the nano enabled products. Moreover, in the last years, the use of new advanced methodologies (NAMs) has increasingly gained attention for the use of alternative methods in obtaining relevant information on NMs hazard and risk. Considering the SSbD and the NAMs frameworks, within the ASINA H2020 project, we developed new NAMs devoted at improving the hazard and risk definition of different Ag and TiO2 NPs. The NAMs are developed considering two air liquid interface exposure systems, the Vitrocell Cloud-α and the Cultex Compact module and the relevant steps to obtain reproducible exposures are described. The new NAMs build on the integration of environmental monitoring campaigns at nano-coating production sites, allowing the quantification by the multiple-path particle dosimetry (MPPD) model of the expected lung deposited dose in occupational settings. Starting from this information, laboratory exposures to the aerosolized NPs are performed by using air liquid interface exposure equipment and human alveolar cells (epithelial cells and macrophages), replicating the doses of exposure estimated in workers by MPPD. Preliminary results on cell viability and inflammatory responses are reported. The proposed NAMs may represent possible future reference procedures for assessing the NPs inhalation toxicology, supporting risk assessment at real exposure doses.

Diesel exhaust particulate emissions and in vitro toxicity from Euro 3 and Euro 6 vehicles.

Incomplete combustion processes in diesel engines produce particulate matter (PM) that significantly contributes to air pollution. Currently, there remains a knowledge gap in relation to the physical and chemical characteristics and also the biological reactivity of the PM emitted from old- and new-generation diesel vehicles. In this study, the emissions from a Euro 3 diesel vehicle were compared to those from a Euro 6 car during the regeneration of a diesel particulate filter (DPF). Different driving cycles were used to collect two types of diesel exhaust particles (DEPs). The particle size distribution was monitored using an engine exhaust particle sizer spectrometer and an electrical low-pressure impactor. Although the Euro 6 vehicle emitted particulates only during DPF regeneration that primarily occurs for a few minutes at high speeds, such emissions are characterized by a higher number of ultrafine particles (<0.1 µm) compared to those from the Euro 3 diesel vehicle. The emitted particles possess different characteristics. For example, Euro 6 DEPs exhibit a lower PAH content than do Euro 3 samples; however, they are enriched in metals that were poorly detected or undetected in Euro 3 emissions. The biological effects of the two DEPs were investigated in human bronchial BEAS-2B cells exposed to 50 µg/mL of PM (corresponding to 5.2 µg/cm2), and the results revealed that Euro 3 DEPs activated the typical inflammatory and pro-carcinogenic pathways induced by combustion-derived particles, while Euro 6 DEPs were less effective in regard to activating such biological responses. Although further investigations are required, it is evident that the different in vitro effects elicited by Euro 3 and Euro 6 DEPs can be correlated with the variable chemical compositions (metals and PAHs) of the emitted particles that play a pivotal role in the inflammatory and carcinogenic potential of airborne PM.

Safety Assessment of Polypyrrole Nanoparticles and Spray-Coated Textiles.

Polypyrrole (PPy) nanoparticles (NPs) are used for the coating of materials, such as textiles, with biomedical applications, including wound care and tissue engineering, but they are also promising antibacterial agents. In this work, PPy NPs were used for the spray-coating of textiles with antimicrobial properties. The functional properties of the materials were verified, and their safety was evaluated. Two main exposure scenarios for humans were identified: inhalation of PPy NPs during spray (manufacturing) and direct skin contact with NPs-coated fabrics (use). Thus, the toxicity properties of PPy NPs and PPy-coated textiles were assessed by using in vitro models representative of the lung and the skin. The results from the materials' characterization showed the stability of both the PPy NP suspension and the textile coating, even after washing cycles and extraction in artificial sweat. Data from an in vitro model of the air-blood barrier showed the low toxicity of these NPs, with no alteration of cell viability and functionality observed. The skin toxicity of PPy NPs and the coated textiles was assessed on a reconstructed human epidermis model following OECD 431 and 439 guidelines. PPy NPs proved to be non-corrosive at the tested conditions, as well as non-irritant after extraction in artificial sweat at two different pH conditions. The obtained data suggest that PPy NPs are safe NMs in applications for textile coating.

Combustion-derived particles from biomass sources differently promote epithelial-to-mesenchymal transition on A549 cells.

Combustion-derived particles (CDPs), due to the presence in their composition of several toxic and carcinogenic chemical compounds, such as polycyclic aromatic hydrocarbons (PAHs) and metals, are linked to several respiratory diseases, including lung cancer. Epithelial-to-mesenchymal transition (EMT) is a crucial step in lung cancer progression, involving several morphological and phenotypical changes. The study aims to investigate how exposure to CDPs from different biomass sources might be involved in cancer development, focusing mainly on the effects linked to EMT and invasion on human A549 lung cells. Biomass combustion-derived particles (BCDPs) were collected from a stove fuelled with pellet, charcoal or wood, respectively. A time course and dose response evaluation on cell viability and pro-inflammatory response was performed to select the optimal conditions for EMT-related studies. A significant release of IL-8 was found after 72 h of exposure to 2.5 µg/cm2 BCDPs. The EMT activation was then examined by evaluating the expression of some typical markers, such as E-cadherin and N-cadherin, and the possible enhanced migration and invasiveness. Sub-acute exposure revealed that BCDPs differentially modulated cell viability, migration and invasion, as well as the expression of proteins linked to EMT. Results showed a reduction in the epithelial marker E-cadherin and a parallel increase in the mesenchymal markers N-cadherin, mainly after exposure to charcoal and wood. Migration and invasion were also increased. In conclusion, our results suggest that BCDPs with a higher content of organic compounds (e.g. PAHs) in their chemical composition might play a crucial role in inducing pro-carcinogenic effects on epithelial cells.

Transcriptional profiling of human bronchial epithelial cell BEAS-2B exposed to diesel and biomass ultrafine particles.

BACKGROUND: Emissions from diesel vehicles and biomass burning are the principal sources of primary ultrafine particles (UFP). The exposure to UFP has been associated to cardiovascular and pulmonary diseases, including lung cancer. Although many aspects of the toxicology of ambient particulate matter (PM) have been unraveled, the molecular mechanisms activated in human cells by the exposure to UFP are still poorly understood. Here, we present an RNA-seq time-course experiment (five time point after single dose exposure) used to investigate the differential and temporal changes induced in the gene expression of human bronchial epithelial cells (BEAS-2B) by the exposure to UFP generated from diesel and biomass combustion. A combination of different bioinformatics tools (EdgeR, next-maSigPro and reactome FI app-Cytoscape and prioritization strategies) facilitated the analyses the temporal transcriptional pattern, functional gene set enrichment and gene networks related to cellular response to UFP particles. RESULTS: The bioinformatics analysis of transcriptional data reveals that the two different UFP induce, since the earliest time points, different transcriptional dynamics resulting in the activation of specific genes. The functional enrichment of differentially expressed genes indicates that the exposure to diesel UFP induces the activation of genes involved in TNFα signaling via NF-kB and inflammatory response, and hypoxia. Conversely, the exposure to ultrafine particles from biomass determines less distinct modifications of the gene expression profiles. Diesel UFP exposure induces the secretion of biomarkers associated to inflammation (CCXL2, EPGN, GREM1, IL1A, IL1B, IL6, IL24, EREG, VEGF) and transcription factors (as NFE2L2, MAFF, HES1, FOSL1, TGIF1) relevant for cardiovascular and lung disease. By means of network reconstruction, four genes (STAT3, HIF1a, NFKB1, KRAS) have emerged as major regulators of transcriptional response of bronchial epithelial cells exposed to diesel exhaust. CONCLUSIONS: Overall, this work highlights modifications of the transcriptional landscape in human bronchial cells exposed to UFP and sheds new lights on possible mechanisms by means of which UFP acts as a carcinogen and harmful factor for human health.

Lung Toxicity of Condensed Aerosol from E-CIG Liquids: Influence of the Flavor and the In Vitro Model Used.

The diffusion of e-cigarette (e-CIG) opens a great scientific and regulatory debate about its safety. The huge number of commercialized devices, e-liquids with almost infinite chemical formulations and the growing market demand for a rapid and efficient toxicity screen system that is able to test all of these references and related aerosols. A consensus on the best protocols for the e-CIG safety assessment is still far to be achieved, since the huge number of variables characterizing these products (e.g., flavoring type and concentration, nicotine concentration, type of the device, including the battery and the atomizer). This suggests that more experimental evidences are needed to support the regulatory frameworks. The present study aims to contribute in this field by testing the effects of condensed aerosols (CAs) from three main e-liquid categories (tobacco, mint, and cinnamon as food-related flavor), with (18 mg/mL) or without nicotine. Two in vitro models, represented by a monoculture of human epithelial alveolar cells and a three-dimensional (3D) co-culture of alveolar and lung microvascular endothelial cells were used. Cell viability, pro-inflammatory cytokines release and alveolar-blood barrier (ABB) integrity were investigated as inhalation toxicity endpoints. Results showed that nicotine itself had almost no influence on the modulation of the toxicity response, while flavor composition did have. The cell viability was significantly decreased in monoculture and ABB after exposure to the mints and cinnamon CAs. The barrier integrity was significantly affected in the ABB after exposure to cytotoxic CAs. With the exception of the significant IL-8 release in the monoculture after Cinnamon exposure, no increase of inflammatory cytokines (IL-8 and MCP-1) release was observed. These findings point out that multiple assays with different in vitro models are able to discriminate the acute inhalation toxicity of CAs from liquids with different flavors, providing the companies and regulatory bodies with useful tools for the preliminary screening of marketable products.

The role of IL-6 released from pulmonary epithelial cells in diesel UFP-induced endothelial activation.

Diesel exhaust particles (DEP) and their ultrafine fraction (UFP) are known to induce cardiovascular effects in exposed subjects. The mechanisms leading to these outcomes are still under investigation, but the activation of respiratory endothelium is likely to be involved. Particles translocation through the air-blood barrier and the release of mediators from the exposed epithelium have been suggested to participate in the process. Here we used a conditioned media in vitro model to investigate the role of epithelial-released mediators in the endothelial cells activation. Diesel UFP were sampled from a Euro 4 vehicle run over a chassis dyno and lung epithelial BEAS-2B cells were exposed for 20 h (dose 5 µg/cm2). The exposure media were collected and used for endothelial HPMEC-ST1.6R cells treatment for 24 h. The processes related to oxidative stress and inflammation were investigated in the epithelial cells, accordingly to the present knowledge on DEP toxicity. The release of IL-6 and VEGF was significantly augmented in diesel exposed cells. In endothelial cells, VCAM-1 and ICAM-1 adhesion molecules levels were increased after exposure to the conditioned media. By interfering with IL-6 binding to its endothelial receptor, we demonstrate the role of this interleukin in inducing the endothelial response.

Impact of zinc oxide nanoparticles on an in vitro model of the human air-blood barrier.

The inhalation of zinc oxide nanoparticles (nZnO) may induce systemic diseases, damages to the alveolar epithelium and inflammatory response to endothelial cells. In this work the use of an in vitro air-blood barrier (ABB) model provided a tool to elucidate the biological mechanisms underlying the potential effects of inhaled nanoparticles (NPs). The ABB model used is composed of a Transwell co-culture of a lung epithelial cell line (NCI-H441) and an immortalized pulmonary microvascular endothelial cell line (HPMEC-ST1.6R). In addition, a tri-culture model was developed by adding monocytes (THP-1) on the basal compartment of the inserts. These models have been set up to analyse the importance of the interplay among the different cell types on various responses after nZnO exposure: inflammation, endothelial damage and modulation of the immune system. The barrier integrity was assessed by measuring the transepithelial electrical resistance (TEER); the pro-inflammatory and immune cells responses were analysed by ELISA. The results have evidenced that nZnO do not affect the barrier integrity, since no TEER reduction was measured after 24h of exposure, but an activation of endothelial cells, which released pro-inflammatory mediators (IL-6, IL-8), and endothelial dysfunction markers (sICAM-1 and sVCAM-1) were induced. These results confirm that apical exposure to NPs promote endothelium activation. The in vitro-ABB model here used is thus a useful tool able to evidence the interaction between lung epithelium and endothelium in inducing biological response, and the role of endothelium dysfunction following NPs inhalation.

Physico-chemical properties and biological effects of diesel and biomass particles.

Diesel combustion and solid biomass burning are the major sources of ultrafine particles (UFP) in urbanized areas. Cardiovascular and pulmonary diseases, including lung cancer, are possible outcomes of combustion particles exposure, but differences in particles properties seem to influence their biological effects. Here the physico-chemical properties and biological effects of diesel and biomass particles, produced under controlled laboratory conditions, have been characterized. Diesel UFP were sampled from a Euro 4 light duty vehicle without DPF fuelled by commercial diesel and run over a chassis dyno. Biomass UFP were collected from a modern automatic 25 kW boiler propelled by prime quality spruce pellet. Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) images of both diesel and biomass samples showed aggregates of soot particles, but in biomass samples ash particles were also present. Chemical characterization showed that metals and PAHs total content was higher in diesel samples compared to biomass ones. Human bronchial epithelial (HBEC3) cells were exposed to particles for up to 2 weeks. Changes in the expression of genes involved in xenobiotic metabolism were observed after exposure to both UFP already after 24 h. However, only diesel particles modulated the expression of genes involved in inflammation, oxidative stress and epithelial-to-mesenchymal transition (EMT), increased the release of inflammatory mediators and caused phenotypical alterations, mostly after two weeks of exposure. These results show that diesel UFP affected cellular processes involved in lung and cardiovascular diseases and cancer. Biomass particles exerted low biological activity compared to diesel UFP. This evidence emphasizes that the study of different emission sources contribution to ambient PM toxicity may have a fundamental role in the development of more effective strategies for air quality improvement.

Release of IL-1 ß triggered by Milan summer PM10: molecular pathways involved in the cytokine release.

Particulate matter (PM) exposure is related to pulmonary and cardiovascular diseases, with increased inflammatory status. The release of the proinflammatory interleukin- (IL-) 1ß, is controlled by a dual pathway, the formation of inactive pro-IL-1ß, through Toll-like receptors (TLRs) activation, and its cleavage by NLRP3 inflammasome. THP-1-derived macrophages were exposed for 6 h to 2.5 µg/cm(2) of Milan PM10, and the potential to promote IL-1ß release by binding TLRs and activating NLRP3 has been examined. Summer PM10, induced a marked IL-1ß response in the absence of LPS priming (50-fold increase compared to unexposed cells), which was reduced by caspase-1 inhibition (91% of inhibition respect summer PM10-treated cells) and by TLR-2 and TLR-4 inhibitors (66% and 53% of inhibition, resp.). Furthermore, summer PM10 increased the number of early endosomes, and oxidative stress inhibition nearly abolished PM10-induced IL-1ß response (90% of inhibition). These findings suggest that summer PM10 contains constituents both related to the activation of membrane TLRs and activation of the inflammasome NLPR3 and that TLRs activation is of pivotal importance for the magnitude of the response. ROS formation seems important for PM10-induced IL-1ß response, but further investigations are needed to elucidate the molecular pathway by which this effect is mediated.

Effect of nanoparticles and environmental particles on a cocultures model of the air-blood barrier.

Exposure to engineered nanoparticles (NPs) and to ambient particles (PM) has increased significantly. During the last decades the application of nano-objects to daily-life goods and the emissions produced in highly urbanized cities have considerably augmented. As a consequence, the understanding of the possible effects of NPs and PM on human respiratory system and particularly on the air-blood barrier (ABB) has become of primary interest. The crosstalk between lung epithelial cells and underlying endothelial cells is indeed essential in determining the effects of inhaled particles. Here we report the effects of metal oxides NPs (CuO and TiO2) and of PM on an in vitro model of the ABB constituted by the type II epithelial cell line (NCI-H441) and the endothelial one (HPMEC-ST1.6R). The results demonstrate that apical exposure of alveolar cells induces significant modulation of proinflammatory proteins also in endothelial cells.