Alexandra Noël

Effects of inhaled nano-TiO2 aerosols showing two distinct agglomeration states on rat lungs

Nano-aerosols composed of large agglomerates (LA) (>100nm) are more likely to promote pulmonary clearance via macrophages phagocytosis. Small agglomerates (SA) (<100nm) seem to escape this first defense mechanism and are more likely to interact directly with biological material. These different mechanisms can influence pulmonary toxicity. This hypothesis was evaluated by comparing the relative pulmonary toxicity induced by aerosolized nano-TiO(2) showing two different agglomeration states: SA (<100nm) and LA (>100nm) at mass concentrations of 2 or 7mg/m(3). Groups of Fisher 344 male rats were nose-only exposed for 6h. The median number aerodynamic diameters were 30 and 185nm at 2mg/m(3), and 31 and 194nm at 7mg/m(3). We found in rats bronchoalveolar lavage fluids (BALF) a significant 2.1-fold increase in the number of neutrophils (p<0.05) in the group exposed to the 7mg/m(3) LA nano-aerosol suggesting a mild inflammatory response. Rats exposed to the 7mg/m(3) SA nano-aerosol showed a 1.8-fold increase in LDH activity and 8-isoprostane concentration in BALF, providing evidence for cytotoxic and oxidative stress effects. Our results indicate that biological responses to nanoparticles (NP) might depend on the dimension and concentration of NP agglomerates.

Rat pulmonary responses to inhaled nano-TiO2: effect of primary particle size and agglomeration state

BackgroundThe exact role of primary nanoparticle (NP) size and their degree of agglomeration in aerosols on the determination of pulmonary effects is still poorly understood. Smaller NP are thought to have greater biological reactivity, but their level of agglomeration in an aerosol may also have an impact on pulmonary response. The aim of this study was to investigate the role of primary NP size and the agglomeration state in aerosols, using well-characterized TiO2 NP, on their relative pulmonary toxicity, through inflammatory, cytotoxic and oxidative stress effects in Fisher 344 male rats.MethodsThree different sizes of TiO2 NP, i.e., 5, 10–30 or 50 nm, were inhaled as small (SA) (< 100 nm) or large agglomerates (LA) (> 100 nm) at 20 mg/m3 for 6 hours.ResultsCompared to the controls, bronchoalveolar lavage fluids (BALF) showed that LA aerosols induced an acute inflammatory response, characterized by a significant increase in the number of neutrophils, while SA aerosols produced significant oxidative stress damages and cytotoxicity. Data also demonstrate that for an agglomeration state smaller than 100 nm, the 5 nm particles caused a significant increase in cytotoxic effects compared to controls (assessed by an increase in LDH activity), while oxidative damage measured by 8-isoprostane concentration was less when compared to 10–30 and 50 nm particles. In both SA and LA aerosols, the 10–30 nm TiO2 NP size induced the most pronounced pro-inflammatory effects compared to controls.ConclusionsOverall, this study showed that initial NP size and agglomeration state are key determinants of nano-TiO2 lung inflammatory reaction, cytotoxic and oxidative stress induced effects.

Impact of emerging pollutants on pulmonary inflammation in asthmatic rats: ethanol vapors and agglomerated TiO2 nanoparticles

Context: Titanium dioxide nanoparticles (nano-TiO2) and ethanol vapors are air contaminants with increasing importance. The presence of a pathological pulmonary condition, such as asthma, may increase lung susceptibility to such contaminants. Objective: This study aimed to investigate if exposure to inhaled ethanol vapors or nano-TiO2 can modulate the rat pulmonary inflammatory response resulting from an allergic asthmatic reaction. Materials and methods: Brown Norway rats were sensitized (sc) and challenged (15 min inhalation, 14 days later) with chicken egg ovalbumin (OVA). Leukocytes were counted in bronchoalveolar lavages (BAL) performed at 6, 24, 36, 48 and 72 h following the challenge and either after ethanol exposures (3000 ppm, 6 h/day, daily) or at 48 h (peak inflammation) for nano-TiO2 exposures (9.35 mg/m3 aerosol for 6 and 42 h after the OVA challenge). For the nano-TiO2 exposures, plasma and BAL cytokines were measured and lung histological analyzes were performed. Results: Exposure to ethanol did not significantly affect BAL leukocytes after OVA challenge. Exposure to nano-TiO2 significantly decreased BAL leukocytes compared to OVA-challenged controls. Plasma and BAL IL-4, IL-6, and INF-γ levels were also decreased in the nano-TiO2 group. Discussion: While ethanol vapors do not modify the pulmonary inflammation in rats during an asthmatic response, a surprising protective effect for agglomerated nano-TiO2 was observed. A putative mechanistic basis involving a decrease in the Th2 response caused by OVA is proposed. Conclusion: Allergic pulmonary inflammation is not up-regulated by inhalation of the pollutants ethanol and nano-TiO2. On the contrary, nano-TiO2 decreases lung inflammation in asthmatic rats.

Generating Nano-Aerosols from TiO2 (5 nm) Nanoparticles Showing Different Agglomeration States. Application to Toxicological Studies

Agglomeration of nanoparticles (NP) is a key factor in the generation of aerosols from nano-powders and may represent an important parameter to consider in toxicological studies. For this reason, the characterization of NP aerosols (e.g., concentration, size, and structure of agglomerates) is a critical step in the determination of the relationship between exposure and effects. The aim of this study was to generate and characterize aerosols composed of TiO2 (5 nm) NP showing different agglomeration states. Two concentrations were tested: 2 and 7 mg/m3. Stable mass concentrations over 6 hr were successfully generated by a wet method using Collison and Delavan nebulizers that resulted in aerosols composed of smaller agglomerates (<100 nm), while aerosols composed of larger agglomerates (>100 nm) were obtained by dry generation techniques using either a Palas dust feeder or a Fluidized Bed. Particle size distributions in the aerosols were determined by an electrical low pressure impactor. Median number aerodynamic diameters corresponding to the aerosol with smaller and larger agglomerates were 30 and 185 nm, respectively, for the 2 mg/m3 concentration, and 31 and 194 nm for the 7 mg/m3 experiment. Image analysis by transmission electron microscopy showed the presence of compact or agglomerates with void spaces in the different nano-aerosols. These characterized nano-aerosols will be used in further experiments to study the influence of agglomerate size on NP toxicity.

Impact of two particle measurement techniques on the determination of N95 class respirator filtration performance against ultrafine particles.

The purpose of this experimental study was to compare two different particle measurement devices; an Electrical Low Pressure Impactor (ELPI) and a Scanning Mobility Particle Sizer (SMPS), to measure the number concentration and the size distribution of NaCl salt aerosols to determine the collection efficiency of filtering respirators against poly disperse aerosols. Tests were performed on NIOSH approved N95 filtering face-piece respirators (FFR), sealed on a manikin head. Ultrafine particles found in the aerosols were also collected and observed by transmission electron microscopy (TEM). According to the results, there is a systematic difference for the particle size distribution measured by the SMPS and the ELPI. It is largely attributed to the difference in the measurement techniques. However, in spite of these discrepancies, reasonably similar trends were found for the number concentration with both measuring instruments. The particle penetration, calculated based on mobility and aerodynamic diameters, never exceeded 5% for any size range measured at constant flow rate of 85 L/min. Also, the most penetrating particle size (MPPS), with the lowest filtration efficiency, would occur at a similar ultrafine size range <100 nm. With the ELPI, the MPPS was at 70 nm aerodynamic diameter, whereas it occurred at 40 nm mobility diameter with the SMPS.

Assessment of the Contribution of Electron Microscopy to Nanoparticle Characterization Sampled with Two Cascade Impactors

This study assessed the contribution of electron microscopy to the characterization of nanoparticles and compared the degree of variability in sizes observed within each stage when sampled by two cascade impactors: an Electrical Low Pressure Impactor (ELPI) and a Micro-Orifice Uniform Deposit Impactor (MOUDI). A TiO2 nanoparticle (5 nm) suspension was aerosolized in an inhalation chamber. Nanoparticles sampled by the impactors were collected on aluminum substrates or TEM carbon-coated copper grids using templates, specifically designed in our laboratories, for scanning and transmission electron microscopy (SEM, TEM) analysis, respectively. Nanoparticles were characterized using both SEM and TEM. Three different types of diameters (inner, outer, and circular) were measured by image analysis based on count and volume, for each impactor stage. Electron microscopy, especially TEM, is well suited for the characterization of nanoparticles. The MOUDI, probably because of the rotation of its collection stages, which can minimize the resuspension of particles, gave more stable results and smaller geometric standard deviations per stage. Our data suggest that the best approach to estimate particle size by electron microscopy would rely on geometric means of measured circular diameters. Overall, the most reliable data were provided by the MOUDI and the TEM sampling technique on carbon-coated copper grids for this specific experiment. This study indicates interesting findings related to the assessment of impactors combined with electron microscopy for nanoparticle characterization. For future research, since cascade impactors are extensively used to characterize nano-aerosol exposure scenarios, high-performance field emission scanning electron microscopy (FESEM) should also be considered.

In utero exposure to second-hand smoke activates pro-asthmatic and oncogenic miRNAs in adult asthmatic mice.

Exposures to environmental pollutants contribute to dysregulated microRNA (miRNA) expression profiles, which have been implicated in various diseases. Previously, we reported aggravated asthmatic responses in ovalbumin (OVA)‐challenged adult mice that had been exposed in utero to second‐hand smoke (SHS). Whether in utero SHS exposure dysregulates miRNA expression patterns in the adult asthma model has not been investigated. Pregnant BALB/c mice were exposed (days 6–19 of pregnancy) to SHS (10 mg/m3) or HEPA‐filtered air. All offspring were sensitized and challenged with OVA (19–23 weeks) before sacrifice. RNA samples extracted from lung homogenates, were subjected to RNA sequencing (RNA‐seq). RNA‐seq identified nine miRNAs that were most significantly up‐regulated by in utero SHS exposure. Among these nine, miR‐155‐5p, miR‐21‐3p, and miR‐18a‐5p were also highly correlated with pro‐asthmatic Th2 cytokine levels in bronchoalveolar lavage fluid. Further analysis indicated that these up‐regulated miRNAs shared common chromosome locations, particularly Chr 11C, with pro‐asthmatic genes. These three miRNAs have also been characterized as oncogenic miRNAs (oncomirs). We cross‐referenced miRNA‐mRNA expression profiles and identified 16 tumor suppressor genes that were down‐regulated in the in utero‐exposed offspring and that are predicted targets of the up‐regulated oncomirs. In conclusion, in utero SHS exposure activates pro‐asthmatic genes and miRNAs, which colocalize at specific chromosome locations, in OVA‐challenged adult mice. The oncogenic characteristics of the miRNAs and putative miRNA‐mRNA regulatory networks suggest that the synergistic effect of in utero SHS exposure and certain adult irritants may promote an oncogenic milieu in mouse lungs via inhibition of miRNA‐regulated tumor suppressor genes. Environ. Mol. Mutagen. 57:190–199, 2016.

Characterization of Aerosols of Titanium Dioxide Nanoparticles Following Three Generation Methods Using an Optimized Aerosolization System Designed for Experimental Inhalation Studies

Nanoparticles (NPs) can be released in the air in work settings, but various factors influence the exposure of workers. Controlled inhalation experiments can thus be conducted in an attempt to reproduce real-life exposure conditions and assess inhalation toxicology. Methods exist to generate aerosols, but it remains difficult to obtain nano-sized and stable aerosols suitable for inhalation experiments. The goal of this work was to characterize aerosols of titanium dioxide (TiO2) NPs, generated using a novel inhalation system equipped with three types of generators—a wet collision jet nebulizer, a dry dust jet and an electrospray aerosolizer—with the aim of producing stable aerosols with a nano-diameter average (<100 nm) and monodispersed distribution for future rodent exposures and toxicological studies. Results showed the ability of the three generation systems to provide good and stable dispersions of NPs, applicable for acute (continuous up to 8 h) and repeated (21-day) exposures. In all cases, the generated aerosols were composed mainly of small aggregates/agglomerates (average diameter <100 nm) with the electrospray producing the finest (average diameter of 70–75 mm) and least concentrated aerosols (between 0.150 and 2.5 mg/m3). The dust jet was able to produce concentrations varying from 1.5 to 150 mg/m3, and hence, the most highly concentrated aerosols. The nebulizer collision jet aerosolizer was the most versatile generator, producing both low (0.5 mg/m3) and relatively high concentrations (30 mg/m3). The three optimized generators appeared suited for possible toxicological studies of inhaled NPs.

Mass or total surface area with aerosol size distribution as exposure metrics for inflammatory, cytotoxic and oxidative lung responses in rats exposed to titanium dioxide nanoparticles

There is currently no consensus on the best exposure metric(s) for expressing nanoparticle (NP) dose. Although surface area has been extensively studied for inflammatory responses, it has not been as thoroughly validated for cytotoxicity or oxidative stress effects. Since inhaled NPs deposit and interact with lung cells based on agglomerate size, we hypothesize that mass concentration combined with aerosol size distribution is suitable for NP risk assessment. The objective of this study was to evaluate different exposure metrics for inhaled 5 nm titanium dioxide aerosols composed of small (SA < 100 nm) or large (LA > 100 nm) agglomerates at 2, 7, and 20 mg/m3 on rat lung inflammatory, cytotoxicity, and oxidative stress responses. We found a significant positive correlation (r = 0.98, p < 0.01) with the inflammatory reaction, measured by the number of neutrophils and the mass concentration when considering all six (SA + LA) aerosols. This correlation was similar (r = 0.87) for total surface area. Regarding cytotoxicity and oxidative stress responses, measured by lactate dehydrogenase and 8-isoprostane, respectively, and mass or total surface area as an exposure metric, we observed significant positive correlations only with SA aerosols for both the mass concentration and size distribution (r > 0.91, p < 0.01), as well as for the total surface area (r > 0.97, p < 0.01). These data show that mass or total surface area concentrations alone are insufficient to adequately predict oxidant and cytotoxic pulmonary effects. Overall, our study indicates that considering NP size distribution along with mass or total surface area concentrations contributes to a more mechanistic discrimination of pulmonary responses to NP exposure.

Early Postnatal Secondhand Smoke Exposure Disrupts Bacterial Clearance and Abolishes Immune Responses in Muco-Obstructive Lung Disease

Secondhand smoke (SHS) exposure has been linked to the worsening of ongoing lung diseases. However, whether SHS exposure affects the manifestation and natural history of imminent pediatric muco-obstructive airway diseases such as cystic fibrosis remains unclear. To address these questions, we exposed Scnn1b transgenic (Scnn1b-Tg+) mice to SHS from postnatal day (PND) 3–21 and lung phenotypes were examined at PND22. Although a majority of filtered air (FA)-exposed Scnn1b-Tg+ (FA-Tg+) mice successfully cleared spontaneous bacterial infections by PND22, the SHS-exposed Scnn1b-Tg+ (SHS-Tg+) mice failed to resolve these infections. This defect was associated with suppressed antibacterial defenses, i.e., phagocyte recruitment, IgA secretion, and Muc5b expression. Whereas the FA-Tg+ mice exhibited marked mucus obstruction and Th2 responses, SHS-Tg+ mice displayed a dramatic suppression of these responses. Mechanistically, downregulated expression of IL-33, a stimulator of type II innate lymphoid cells, in lung epithelial cells was associated with suppression of neutrophil recruitment, IgA secretions, Th2 responses, and delayed bacterial clearance in SHS-Tg+ mice. Cessation of SHS exposure for 21 d restored previously suppressed responses, including phagocyte recruitment, IgA secretion, and mucous cell metaplasia. However, in contrast with FA-Tg+ mice, the SHS-Tg+ mice had pronounced epithelial necrosis, alveolar space consolidation, and lymphoid hyperplasia; indicating lagged unfavorable effects of early postnatal SHS exposure in later life. Collectively, our data show that early postnatal SHS exposure reversibly suppresses IL-33 levels in airspaces which, in turn, results in reduced neutrophil recruitment and diminished Th2 response. Our data indicate that household smoking may predispose neonates with muco-obstructive lung disease to bacterial exacerbations.