In Vitro Toxicity of Industrially Relevant Engineered Nanoparticles in Human Alveolar Epithelial Cells: Air-Liquid Interface versus Submerged Cultures

Diverse industries have already incorporated within their production processes engineered nanoparticles (ENP), increasing the potential risk of worker inhalation exposure. In vitro models have been widely used to investigate ENP toxicity. Air–liquid interface (ALI) cell cultures have been emerging as a valuable alternative to submerged cultures as they are more representative of the inhalation exposure to airborne nano-sized particles. We compared the in vitro toxicity of four ENP used as raw materials in the advanced ceramics sector in human alveolar epithelial-like cells cultured under submerged or ALI conditions. Submerged cultures were exposed to ENP liquid suspensions or to aerosolised ENP at ALI. Toxicity was assessed by determining LDH release, WST-1 metabolisation and DNA damage. Overall, cells were more sensitive to ENP cytotoxic effects when cultured and exposed under ALI. No significant cytotoxicity was observed after 24 h exposure to ENP liquid suspensions, although aerosolised ENP clearly affected cell viability and LDH release. In general, all ENP increased primary DNA damage regardless of the exposure mode, where an increase in DNA strand-breaks was only detected under submerged conditions. Our data show that at relevant occupational concentrations, the selected ENP exert mild toxicity to alveolar epithelial cells and exposure at ALI might be the most suitable choice when assessing ENP toxicity in respiratory models under realistic exposure conditions.This research was funded by CERASAFE (www.cerasafe.eu; accessed on 26 October 2021), with the support of ERA-NET SIINN (project id:16) and the Portuguese Foundation for Science and Technology (FCT; SIINN/0004/2014). This work was also supported by the NanoBioBarriers project (PTDC/MED-TOX/31162/2017), co-financed by the Operational Program for Competitiveness and Internationalization (POCI) through European Regional Development Funds (FEDER/FNR) and FCT; Spanish Ministry of Science and Innovation (projects PCIN-2015-173-C02-01 and CEX2018-000794-S-Severo Ochoa), and by the Romanian National Authority for Scientific Research and Innovation (CCCDI-UEFISCDI, project number 29/2016 within PNCDI III). M.J. Bessa (SFRH/BD/120646/2016) and F. Brandão (SFRH/BD/101060/2014) are recipients of FCT PhD scholarships under the framework of Human Capital Operating Program (POCH) and European Union funding. The Doctoral Program in Biomedical Sciences, of the ICBAS—University of Porto, offered additional funds. S. Fraga thanks FCT for funding through program DL 57/2016–Norma transitória (Ref. DL-57/INSA-06/2018). Thanks are also due to FCT/MCTES for the financial support to EPIUnit (info:eu-repo/grantAgreement/FCT/6817 - DCRRNI ID/UIDB/04750/2020/PT)

Unveiling the Toxicity of Fine and Nano-Sized Airborne Particles Generated from Industrial Thermal Spraying Processes in Human Alveolar Epithelial Cells

High-energy industrial processes have been associated with particle release into workplace air that can adversely affect workers' health. The present study assessed the toxicity of incidental fine (PGFP) and nanoparticles (PGNP) emitted from atmospheric plasma (APS) and high-velocity oxy-fuel (HVOF) thermal spraying. Lactate dehydrogenase (LDH) release, 2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate (WST-1) metabolisation, intracellular reactive oxygen species (ROS) levels, cell cycle changes, histone H2AX phosphorylation (γ-H2AX) and DNA damage were evaluated in human alveolar epithelial cells at 24 h after exposure. Overall, HVOF particles were the most cytotoxic to human alveolar cells, with cell viability half-maximal inhibitory concentration (IC50) values of 20.18 μg/cm2 and 1.79 μg/cm2 for PGFP and PGNP, respectively. Only the highest tested concentration of APS-PGFP caused a slight decrease in cell viability. Particle uptake, cell cycle arrest at S + G2/M and γ-H2AX augmentation were observed after exposure to all tested particles. However, higher levels of γ-H2AX were found in cells exposed to APS-derived particles (~16%), while cells exposed to HVOF particles exhibited increased levels of oxidative damage (~17% tail intensity) and ROS (~184%). Accordingly, APS and HVOF particles seem to exert their genotoxic effects by different mechanisms, highlighting that the health risks of these process-generated particles at industrial settings should not be underestimated. © 2022 by the authors. Licensee MDPI, Basel, Switzerland.Funding text 1: transitória (Ref. DL-57/INSA-06/2018). Thanks are also due to FCT/MCTES for the financial support to EPIUnit and ITR (UIDB/04750/2020 and LA/P/0064/2020). Informed Consent Statement: Not applicable. Institutional Review Board Statement: Not applicable.; Funding text 2: Funding: This research was funded by the project CERASAFE with the support of ERA-NET SIINN (project id:16) and the Portuguese Foundation for Science and Technology (FCT; SIINN/0004/2014). This work was also supported by the project NanoBioBarriers (PTDC/MED-TOX/31162/2017), cofinanced by the Operational Program for Competitiveness and Internationalization (POCI) through European Regional Development Funds (FEDER/FNR) and FCT and the Spanish Ministry of Sci- ence and Innovation (projects PCIN-2015-173-C02-01 and CEX2018-000794-S-Severo Ochoa). M.J. Bessa (SFRH/BD/120646/2016) and F. Brandão (SFRH/BD/101060/2014) are recipients of FCT PhD scholarships under the framework of the Human Capital Operating Program (POCH) and European Union funding. The Doctoral Program in Biomedical Sciences of the ICBAS—University of Porto Int.J.Mol. Sci. 2022,of23fer, x FOR PEER REVIEWed additional funds. S. Fraga thanks FCT for funding through program DL 57/2016—Norm

In Vitro Toxicity of Industrially Relevant Engineered Nanoparticles in Human Alveolar Epithelial Cells: Air-Liquid Interface versus Submerged Cultures

This article belongs to the Special Issue Engineered Nanomaterials Exposure and Risk Assessment: Occupational Health and SafetyDiverse industries have already incorporated within their production processes engineered nanoparticles (ENP), increasing the potential risk of worker inhalation exposure. In vitro models have been widely used to investigate ENP toxicity. Air-liquid interface (ALI) cell cultures have been emerging as a valuable alternative to submerged cultures as they are more representative of the inhalation exposure to airborne nano-sized particles. We compared the in vitro toxicity of four ENP used as raw materials in the advanced ceramics sector in human alveolar epithelial-like cells cultured under submerged or ALI conditions. Submerged cultures were exposed to ENP liquid suspensions or to aerosolised ENP at ALI. Toxicity was assessed by determining LDH release, WST-1 metabolisation and DNA damage. Overall, cells were more sensitive to ENP cytotoxic effects when cultured and exposed under ALI. No significant cytotoxicity was observed after 24 h exposure to ENP liquid suspensions, although aerosolised ENP clearly affected cell viability and LDH release. In general, all ENP increased primary DNA damage regardless of the exposure mode, where an increase in DNA strand-breaks was only detected under submerged conditions. Our data show that at relevant occupational concentrations, the selected ENP exert mild toxicity to alveolar epithelial cells and exposure at ALI might be the most suitable choice when assessing ENP toxicity in respiratory models under realistic exposure conditions.This research was funded by CERASAFE (www.cerasafe.eu; accessed on 26 October 2021), with the support of ERA-NET SIINN (project id:16) and the Portuguese Foundation for Science and Technology (FCT; SIINN/0004/2014). This work was also supported by the NanoBioBarriers project (PTDC/MED-TOX/31162/2017), co-financed by the Operational Program for Competitiveness and Internationalization (POCI) through European Regional Development Funds (FEDER/FNR) and FCT; Spanish Ministry of Science and Innovation (projects PCIN-2015-173-C02-01 and CEX2018-000794- S-Severo Ochoa), and by the Romanian National Authority for Scientific Research and Innovation (CCCDI-UEFISCDI, project number 29/2016 within PNCDI III). M.J. Bessa (SFRH/BD/120646/2016) and F. Brandão (SFRH/BD/101060/2014) are recipients of FCT PhD scholarships under the framework of Human Capital Operating Program (POCH) and European Union funding. The Doctoral Program in Biomedical Sciences, of the ICBAS—University of Porto, offered additional funds. S. Fraga thanks FCT for funding through program DL 57/2016–Norma transitória (Ref. DL-57/INSA-06/2018). Thanks are also due to FCT/MCTES for the financial support to EPIUnit (UIDB/04750/2020).info:eu-repo/semantics/publishedVersio

In vitro toxicity of industrially relevant engineered nanoparticles in human alveolar epithelial cells: air–liquid interface versus submerged cultures

Diverse industries have already incorporated within their production processes engineered nanoparticles (ENP), increasing the potential risk of worker inhalation exposure. In vitro models have been widely used to investigate ENP toxicity. Air–liquid interface (ALI) cell cultures have been emerging as a valuable alternative to submerged cultures as they are more representative of the inhalation exposure to airborne nano-sized particles. We compared the in vitro toxicity of four ENP used as raw materials in the advanced ceramics sector in human alveolar epithelial-like cells cultured under submerged or ALI conditions. Submerged cultures were exposed to ENP liquid suspensions or to aerosolised ENP at ALI. Toxicity was assessed by determining LDH release, WST-1 metabolisation and DNA damage. Overall, cells were more sensitive to ENP cytotoxic effects when cultured and exposed under ALI. No significant cytotoxicity was observed after 24 h exposure to ENP liquid suspensions, although aerosolised ENP clearly affected cell viability and LDH release. In general, all ENP increased primary DNA damage regardless of the exposure mode, where an increase in DNA strand-breaks was only detected under submerged conditions. Our data show that at relevant occupational concentrations, the selected ENP exert mild toxicity to alveolar epithelial cells and exposure at ALI might be the most suitable choice when assessing ENP toxicity in respiratory models under realistic exposure conditions

Characterizing the Chemical Profile of Incidental Ultrafine Particles for Toxicity Assessment Using an Aerosol Concentrator

Incidental ultrafine particles (UFPs) constitute a key pollutant in industrial workplaces. However, characterizing their chemical properties for exposure and toxicity assessments still remains a challenge. In this work, the performance of an aerosol concentrator (Versatile Aerosol Concentration Enrichment System, VACES) was assessed to simultaneously sample UFPs on filter substrates (for chemical analysis) and as liquid suspensions (for toxicity assessment), in a high UFP concentration scenario. An industrial case study was selected where metal-containing UFPs were emitted during thermal spraying of ceramic coatings. Results evidenced the comparability of the VACES system with online monitors in terms of UFP particle mass (for concentrations up to 95 µg UFP/m3 ) and between filters and liquid suspensions, in terms of particle composition (for concentrations up to 1000 µg/ m3). This supports the applicability of this tool for UFP collection in view of chemical and toxicological characterization for incidental UFPs. In the industrial setting evaluated, results showed that the spraying temperature was a driver of fractionation of metals between UF (<0.2 µm) and fine (0.2– 2.5 µm) particles. Potentially health hazardous metals (Ni, Cr) were enriched in UFPs and depleted in the fine particle fraction. Metals vaporized at high temperatures and concentrated in the UF fraction through nucleation processes. Results evidenced the need to understand incidental particle formation mechanisms due to their direct implications on particle composition and, thus, exposure. It is advisable that personal exposure and subsequent risk assessments in occupational settings should include dedicated metrics to monitor UFPs (especially, incidental).What’s important about this paper: Our work addresses the challenge of characterizing the bulk chemical composition of ultrafine particles in occupational settings, for exposure and toxicity assessments. We tested the performance of an aerosol concentrator (VACES) to simultaneously sample ultrafine particles (UFPs) on filter substrates and as liquid suspensions, in a high UFP concentration scenario. An industrial case study was selected where metal-bearing UFPs were emitted. We report the chemical exposures characterized in the industrial facility, and evidence the comparability of the VACES system with online monitors for UFP particle mass (up to 95 µg UFP/m3) as well as between UFP chemical composition on filters and in suspension. This supports the applicability of this tool for UFP collection in view of chemical and toxicological characterization of exposures to incidental UFPs in workplace settings.Highlights: - The VACES system is a useful tool for UFP sampling in high-concentration settings; - UFP collected simultaneously on filters and in suspension showed good comparability; - UFP chemical profiles were characterized; - Health-hazardous metals Ni and Cr accumulated in UFPs; - Understanding emission mechanisms is key to identifying exposure sources.This work was funded by SIINN ERA-NET (project id: 16), the Spanish MINECO (PCIN-2015-173-C02-01) and the French agency (Region Hauts de France). The Spanish Ministry of Science and Innovation (Project CEX2018-000794-S; Severo Ochoa) and the Generalitat de Catalunya (project number: AGAUR 2017 SGR41) provided support for the indirect costs for the Institute of Environmental Assessment and Water Research (IDAEA-CSIC). We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI).info:eu-repo/semantics/publishedVersio

Particle size distributions and hygroscopic restructuring of ultrafine particles emitted during thermal spraying

We report measurements of the size, concentration, and hygroscopicity of ultrafine particles (UFPs) emitted during thermal spraying of ceramic coatings in an industrial setting. High concentrations (i.e., higher than 106 cm-3) of fractal-like UFPs were measured inside the spraying booths of the facility. The emitted UFPs were found to take up small amounts of water when exposed to elevated relative humidity (RH = 87%) within a Hygroscopic Tandem Differential Mobility Analyzer (HTDMA) system. The hygroscopicity of the sampled particles was distinguishably lower compared to those of the atmospheric background aerosol particles present in the breathing air. UFPs smaller than 90 nm that are produced by the thermal spraying process, exhibit hygroscopic factors less than unity in a systematic way. This behaviour indicates that the particles were irregularly shaped at dry conditions, and that they underwent a shape change (i.e., restructuring) upon humidification inside the HTDMA. The fractal-like structure of processemitted UFPs was further corroborated by Transmission Electron Microscopy (TEM) conducted on samples collected on site at dry conditions.The authors kindly acknowledge TM COMAS (http://www.tmcomas.com) for their committed cooperation. The current work was carried out in the framework of the CERASAFE project (www.cerasafe.eu), with the support of SIINN ERA-NET (project id: 16), and was funded by the Spanish MINECO (PCIN-2015-173-C02-01) and the respective French agency (Region Hauts de France). Partial funding from the “Generalitat de Catalunya” (project number: AGAUR 2017 SGR41) is also acknowledged.Peer reviewe

Particle size distributions and hygroscopic restructuring of ultrafine particles emitted during thermal spraying

We report measurements of the size, concentration, and hygroscopicity of ultrafine particles (UFPs) emitted during thermal spraying of ceramic coatings in an industrial setting. High concentrations (i.e., higher than 106 cm−3) of fractal-like UFPs were measured inside the spraying booths of the facility. The emitted UFPs were found to take up small amounts of water when exposed to elevated relative humidity (RH = 87%) within a Hygroscopic Tandem Differential Mobility Analyzer (HTDMA) system. The hygroscopicity of the sampled particles was distinguishably lower compared to those of the atmospheric background aerosol particles present in the breathing air. UFPs smaller than 90 nm that are produced by the thermal spraying process, exhibit hygroscopic factors less than unity in a systematic way. This behavior indicates that the particles were irregularly shaped at dry conditions, and that they underwent a shape change (i.e., restructuring) upon humidification inside the HTDMA. The fractal-like structure of process-emitted UFPs was further corroborated by Transmission Electron Microscopy (TEM) conducted on samples collected at dry conditions on site.Accepted Author ManuscriptAtmospheric Remote Sensin

Effectiveness of nanoparticle exposure mitigation measures in industrial settings

International audienc

Chemical cross-linking of anatase nanoparticle thin films for enhanced mechanical properties

Titania nanoparticle-based thin films are highly attractive for a vast range of commercial applications. Although their application on polymer-based substrates is particularly appealing, the requirement of low process temperatures results in low mechanical stability. Highly crystalline anatase nanoparticles were used as the building blocks for coatings through a two-stage process. The main benefits of this method, over the more common sol–gel ones, are the relatively low temperature required for the production of metal oxide coatings, allowing the use of polymer-based substrates, and the defined crystallinity of the resulting thin films. Although in several cases moderate temperatures can be utilized for drying the films, the mechanical stability of the respective coatings remains a critical issue. In this contribution, we present a strategy to achieve network formation between TiO₂ nanoparticles in a preformed thin film on the basis of the cross-linking of the functionalized nanoparticles. In the first stage, the nanoparticles were functionalized by dicarboxylic acids, concurrently leading to a stable colloidal dispersion that could be utilized for dip-coating to obtain TiO₂ thin films with high homogeneity and optical transparence. During the second stage, the films were immersed in a solution of a diamine as the linker molecule, to achieve cross-linking between the nanoparticles within the film. It is demonstrated that indeed covalent bonding was realized and functional coatings with significantly enhanced mechanical properties were obtained by our strategy

Workplace Exposure to Nanoparticles during Thermal Spraying of Ceramic Coatings

Thermal spraying is widely used for industrial-scale application of ceramic coatings onto metallic surfaces. The particular process has implications for occupational health, as the high energy process generates high emissions of metal-bearing nanoparticles. Emissions and their impact on exposure were characterized during thermal spraying in a work environment, by monitoring size-resolved number and mass concentrations, lung-deposited surface area, particle morphology, and chemical composition. Along with exposure quantification, the modal analysis of the emissions assisted in distinguishing particles from different sources, while an inhalation model provided evidence regarding the potential deposition of particulate matter on human respiratory system. High particle number (>106 cm-3; 30-40 nm) and mass (60-600 µgPM1 m-3) concentrations were recorded inside the spraying booths, which impacted exposure in the worker area (104-105 cm-3, 40-65 nm; 44-87 µgPM1 m-3). Irregularly-shaped, metal-containing particles (Ni, Cr, W) were sampled from the worker area, as single particles and aggregates (5-200 nm). Energy dispersive X-ray analysis confirmed the presence of particles originated from the coating material, establishing a direct link between the spraying activity and exposure. In particle number count, 90% of the particles were between 26-90 nm. Inhaled dose rates, calculated from the exposure levels, resulted in particle number rates (n˙) between 353 × 106-1024 × 106 min-1, with 70% of deposition occurring in the alveolar region. The effectiveness of personal protective equipment (FPP3 masks) was tested under real working conditions. The proper sealing of the spraying booths was identified as a key element for exposure reduction. This study provides high time-resolved aerosol data which may be valuable for validating indoor aerosol models applied to risk assessment