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Toxicity assessment of industrial engineered and airborne process-generated nanoparticles in a 3D human airway epithelial in vitro model

Publication . Bessa, Maria João; Brandão, Fátima; Fokkens, Paul; Cassee, Flemming R.; Salmatonidis, Apostolos; Viana, Mar; Vulpoi, Adriana; Simon, Simion; Monfort, Eliseo; Teixeira, João Paulo; Fraga, Sónia

The advanced ceramic technology has been pointed out as a potentially relevant case of occupational exposure to nanoparticles (NP). Not only when nanoscale powders are being used for production, but also in the high-temperature processing of ceramic materials there is also a high potential for NP release into the workplace environment. In vitro toxicity of engineered NP (ENP) [antimony tin oxide (Sb2O3•SnO2; ATO); zirconium oxide (ZrO2)], as well as process-generated NP (PGNP), and fine particles (PGFP), was assessed in MucilAir™ cultures at air-liquid interface (ALI). Cultures were exposed during three consecutive days to varying doses of the aerosolized NP. General cytotoxicity [lactate dehydrogenase (LDH) release, WST-1 metabolization], (oxidative) DNA damage, and the levels of pro-inflammatory mediators (IL-8 and MCP-1) were assessed. Data revealed that ENP (5.56 µg ATO/cm2 and 10.98 µg ZrO2/cm2) only caused mild cytotoxicity at early timepoints (24 h), whereas cells seemed to recover quickly since no significant changes in cytotoxicity were observed at late timepoints (72 h). No meaningful effects of the ENP were observed regarding DNA damage and cytokine levels. PGFP affected cell viability at dose levels as low as ∼9 µg/cm2, which was not seen for PGNP. However, exposure to PGNP (∼4.5 µg/cm2) caused an increase in oxidative DNA damage. These results indicated that PGFP and PGNP exhibit higher toxicity potential than ENP in mass per area unit. However, the presence of a mucociliary apparatus, as it occurs in vivo as a defense mechanism, seems to considerably attenuate the observed toxic effects. Our findings highlight the potential hazard associated with exposure to incidental NP in industrial settings.

2021-05Journal article

Restricted access

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

Publication . Bessa, Maria João; Brandão, Fátima; Fokkens, Paul H.B.; Leseman, Daan L.A.C.; Boere, A. John F.; Cassee, Flemming R.; Salmatonidis, Apostolos; Viana, Mar; Monfort, Eliseo; Fraga, Sónia; Teixeira, João Paulo

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.

2022-04-13Journal article

Open access

In vitro acute toxicity of metal-based nanoparticles in human trophoblast BeWo b30 cells

Publication . Pires, J.; Moreira, L.; Teixeira, J.P.; Fraga, S.

Metal nanoparticles (M-NP) are among the most widely used nanomaterials in consumer products available in the market. Thus, human exposure to these nanosized materials is increasing, which raises serious concerns regarding their environmental and human safety. Biological barriers are important lines of defence to xenobiotics, thus expected targets for M-NP. In this regard, special consideration must be given to the placenta that acts as barrier between maternal and the developing fetus. The present study aimed at evaluating in vitro toxicity of different M-NP in a human cell model of placental barrier: trophoblastic (BeWo clone b30) epithelial cells. BeWo b30 cells were exposed for 24 h to varied concentrations (0.8–48 µg/cm2) of M-NP of different chemical composition (Au, Ag and TiO2), primary size (10, 30 and 60 nm), capping (citrate and PEG) and crystal structure (rutile and anatase).

2021-09-24Conference object

Restricted access

Assessing the in vitro toxicity of engineered and airborne nanoceramics: contribution to the safe production and use of nanomaterials in the ceramic industry

Publication . Bessa, Maria João; Fraga, Sónia; Teixeira, João Paulo; Laffon, Blanca Lage

Advanced ceramic technologies have a strong potential for airborne (nano)particle formation and emission, meaning that workers of those industries are at great risk of exposure to these particles. However, toxicological data of these (nano)particles is lacking, particularly for airborne particles released within sectors such as the ceramic industry. To address this relevant topic, the present work aimed to assess the toxicity of occupationally relevant doses of industrially process-generated particles emitted during two industrial thermal spraying technologies [atmospheric plasma spraying (APS) and high velocity oxy-fuel (HVOF)], as well as of four engineered nanoparticles [ENP; tin oxide (SnO2), antimony-tin oxide (ATO; Sb2O3●SnO2), cerium oxide (CeO2) and zirconium oxide (ZrO2)] used as raw materials for ceramics manufacture. Two human respiratory in vitro systems, either conventional alveolar epithelial A549 cultures under submerged or air-liquid interface (ALI) conditions, or advanced three-dimensional (3D) upper airway epithelium (MucilAirTM) cultures at ALI were exposed to the selected particles. Major toxicity endpoints including plasma membrane integrity, metabolic activity, oxidative stress, inflammatory response, and genotoxicity were assessed. Overall, the tested process-generated particles seem to be more toxic compared to the ENP, most likely due to their higher chemical complexity and composition [elevated levels of metallic elements like chromium (Cr) and nickel (Ni)]. Among the two evaluated thermal spraying processes, particles derived from HVOF were more cytotoxic than those emitted from APS. Either fine (PGFP) and ultrafine (PGNP) particles from both spraying processes were able to induce measurable genotoxic effects. While APS particles lead to increased levels of histone 2AX (H2AX) phosphorylation, HVOF particles caused 8-oxo-7,8-dihydroguanine (8-oxo-G) oxidative DNA lesions. ENP were more toxic to human alveolar epithelial cultures when aerosolised than in liquid suspension, particularly ZrO2 NP. On the other hand, advanced MucilAirTM cultures, that better mimic in vivo physiological features, such as the mucociliary defence mechanisms, were quite resistant to both HVOF-derived particles and ENP aerosols. Thus, while 3D human upper airway epithelial cultures exhibited attenuated responses, the conventional A549 cultures were more sensitive to the studied (nano)particles.The present work highlights the hazard of industrially derived (nano)particles, either intentionally used or incidentally released into the workplace air during advanced ceramic processes. Importantly, particles’ physicochemical properties alongside the testing conditions (cell model and type of exposure) played a determinant role in the observed biological responses. These findings reinforce the importance of using physiologically relevant in vitro models in (nano)particle toxicity studies, for better data extrapolation to humans.

2022-07-01Doctoral thesis

Open access