Cellular responses after exposure of lung cell cultures to secondary organic aerosol particles

The scope of this work was to examine in vitro responses of lung cells to secondary organic aerosol (SOA) particles, under realistic ambient air and physiological conditions occurring when particles are inhaled by mammals, using a novel particle deposition chamber. The cell cultures included cell types that are representative for the inner surface of airways and alveoli and are the target cells for inhaled particles. The results demonstrate that an exposure to SOA at ambient-air concentrations of about 10(4) particles/cm(3) for 2 h leads to only moderate cellular responses. There is evidence for (i) cell type specific effects and for (ii) different effects of SOA originating from anthropogenic and biogenic precursors, i.e. 1,3,5-trimethylbenzene (TMB) and alpha-pinene, respectively. There was no indication for cytotoxic effects but for subtle changes in cellular functions that are essential for lung homeostasis. Decreased phagocytic activity was found in human macrophages exposed to SOA from alpha-pinene. Alveolar epithelial wound repair was affected by TMB-SOA exposure, mainly because of altered cell spreading and migration at the edge of the wound. In addition, cellular responses were found to correlate with particle number concentration, as interleukin-8 production was increased in pig explants exposed to TMB-SOA with high particle numbers.

MORTALITY IN THE MEDICARE POPULATION AND CHRONIC EXPOSURE TO FINE PARTICULATE AIR POLLUTION

Prospective cohort studies have provided evidence on longer-term mortality risks of fine particulate matter (PM2.5), but due to their complexity and costs, only a few have been conducted. By linking monitoring data to the U.S. Medicare system by county of residence, we developed a retrospective cohort study, the Medicare Air Pollution Cohort Study (MCAPS), comprising over 20 million enrollees in the 250 largest counties during 2000-2002. We estimated log-linear regression models having as outcome the age-specific mortality rate for each county and as the main predictor, the average level for the study period 2000. Area-level covariates were used to adjust for socio-economic status and smoking. We reported results under several degrees of adjustment for spatial confounding and with stratification into by eastern, central and western counties. We estimated that a 10 µg/m3 increase in PM25 is associated with a 7.6% increase in mortality (95% CI: 4.4 to 10.8%). We found a stronger association in the eastern counties than nationally, with no evidence of an association in western counties. When adjusted for spatial confounding, the estimated log-relative risks drop by 50%. We demonstrated the feasibility of using Medicare data to establish cohorts for follow-up for effects of air pollution. Particulate matter (PM) air pollution is a global public health problem (1). In developing countries, levels of airborne particles still reach concentrations at which serious health consequences are well-documented; in developed countries, recent epidemiologic evidence shows continued adverse effects, even though particle levels have declined in the last two decades (2-6). Increased mortality associated with higher levels of PM air pollution has been of particular concern, giving an imperative for stronger protective regulations (7). Evidence on PM and health comes from studies of acute and chronic adverse effects (6). The London Fog of 1952 provides dramatic evidence of the unacceptable short-term risk of extremely high levels of PM air pollution (8-10); multi-site time-series studies of daily mortality show that far lower levels of particles are still associated with short-term risk (5)(11-13). Cohort studies provide complementary evidence on the longer-term risks of PM air pollution, indicating the extent to which exposure reduces life expectancy. The design of these studies involves follow-up of cohorts for mortality over periods of years to decades and an assessment of mortality risk in association with estimated long-term exposure to air pollution (2-4;14-17). Because of the complexity and costs of such studies, only a small number have been conducted. The most rigorously executed, including the Harvard Six Cities Study and the American Cancer Society’s (ACS) Cancer Prevention Study II, have provided generally consistent evidence for an association of long- term exposure to particulate matter air pollution with increased all-cause and cardio-respiratory mortality (2,4,14,15). Results from these studies have been used in risk assessments conducted for setting the U.S. National Ambient Air Quality Standard (NAAQS) for PM and for estimating the global burden of disease attributable to air pollution (18,19). Additional prospective cohort studies are necessary, however, to confirm associations between long-term exposure to PM and mortality, to broaden the populations studied, and to refine estimates by regions across which particle composition varies. Toward this end, we have used data from the U.S. Medicare system, which covers nearly all persons 65 years of age and older in the United States. We linked Medicare mortality data to (particulate matter less than 2.5 µm in aerodynamic diameter) air pollution monitoring data to create a new retrospective cohort study, the Medicare Air Pollution Cohort Study (MCAPS), consisting of 20 million persons from 250 counties and representing about 50% of the US population of elderly living in urban settings. In this paper, we report on the relationship between longer-term exposure to PM2.5 and mortality risk over the period 2000 to 2002 in the MCAPS.

A novel exposure system for the efficient and controlled deposition of aerosol particles onto cell cultures

Epidemiologic studies have shown correlations between morbidity and particles < or = 2.5 microm generated from pollution processes and manufactured nanoparticles. Thereby nanoparticles seem to play a specific role. The interaction of particles with the lung, the main pathway of undesired particle uptake, is poorly understood. In most studies investigating these interactions in vitro, particle deposition differs greatly from the in vivo situation, causing controversial results. We present a nanoparticle deposition chamber to expose lung cells mimicking closely the particle deposition conditions in the lung. In this new deposition chamber, particles are deposited very efficiently, reproducibly, and uniformly onto the cell culture, a key aspect if cell responses are quantified in respect to the deposited particle number. In situ analyses of the lung cells, e.g., the ciliary beat frequency, indicative of the defense capability of the cells, are complemented by off-line biochemical, physiological, and morphological cell analyses.

A dose-controlled system for air-liquid interface cell exposure and application to zinc oxide nanoparticles

BACKGROUND: Engineered nanoparticles are becoming increasingly ubiquitous and their toxicological effects on human health, as well as on the ecosystem, have become a concern. Since initial contact with nanoparticles occurs at the epithelium in the lungs (or skin, or eyes), in vitro cell studies with nanoparticles require dose-controlled systems for delivery of nanoparticles to epithelial cells cultured at the air-liquid interface. RESULTS: A novel air-liquid interface cell exposure system (ALICE) for nanoparticles in liquids is presented and validated. The ALICE generates a dense cloud of droplets with a vibrating membrane nebulizer and utilizes combined cloud settling and single particle sedimentation for fast (~10 min; entire exposure), repeatable (<12%), low-stress and efficient delivery of nanoparticles, or dissolved substances, to cells cultured at the air-liquid interface. Validation with various types of nanoparticles (Au, ZnO and carbon black nanoparticles) and solutes (such as NaCl) showed that the ALICE provided spatially uniform deposition (<1.6% variability) and had no adverse effect on the viability of a widely used alveolar human epithelial-like cell line (A549). The cell deposited dose can be controlled with a quartz crystal microbalance (QCM) over a dynamic range of at least 0.02-200 mug/cm(2). The cell-specific deposition efficiency is currently limited to 0.072 (7.2% for two commercially available 6-er transwell plates), but a deposition efficiency of up to 0.57 (57%) is possible for better cell coverage of the exposure chamber. Dose-response measurements with ZnO nanoparticles (0.3-8.5 mug/cm(2)) showed significant differences in mRNA expression of pro-inflammatory (IL-8) and oxidative stress (HO-1) markers when comparing submerged and air-liquid interface exposures. Both exposure methods showed no cellular response below 1 mug/cm(2 )ZnO, which indicates that ZnO nanoparticles are not toxic at occupationally allowed exposure levels. CONCLUSION: The ALICE is a useful tool for dose-controlled nanoparticle (or solute) exposure of cells at the air-liquid interface. Significant differences between cellular response after ZnO nanoparticle exposure under submerged and air-liquid interface conditions suggest that pharmaceutical and toxicological studies with inhaled (nano-)particles should be performed under the more realistic air-liquid interface, rather than submerged cell conditions.

Direct combination of nanoparticle fabrication and exposure to lung cell cultures in a closed setup as a method to simulate accidental nanoparticle exposure of humans

The tremendous application potential of nanosized materials stays in sharp contrast to a growing number of critical reports of their potential toxicity. Applications of in vitro methods to assess nanoparticles are severely limited through difficulties in exposing cells of the respiratory tract directly to airborne engineered nanoparticles. We present a completely new approach to expose lung cells to particles generated in situ by flame spray synthesis. Cerium oxide nanoparticles from a single run were produced and simultaneously exposed to the surface of cultured lung cells inside a glovebox. Separately collected samples were used to measure hydrodynamic particle size distribution, shape, and agglomerate morphology. Cell viability was not impaired by the conditions of the glovebox exposure. The tightness of the lung cell monolayer, the mean total lamellar body volume, and the generation of oxidative DNA damage revealed a dose-dependent cellular response to the airborne engineered nanoparticles. The direct combination of production and exposure allows studying particle toxicity in a simple and reproducible way under environmental conditions.

Exposure of silver-nanoparticles and silver-ions to lung cells in vitro at the air-liquid interface

BACKGROUND: Due to its antibacterial properties, silver (Ag) has been used in more consumer products than any other nanomaterial so far. Despite the promising advantages posed by using Ag-nanoparticles (NPs), their interaction with mammalian systems is currently not fully understood. An exposure route via inhalation is of primary concern for humans in an occupational setting. Aim of this study was therefore to investigate the potential adverse effects of aerosolised Ag-NPs using a human epithelial airway barrier model composed of A549, monocyte derived macrophage and dendritic cells cultured in vitro at the air-liquid interface. Cell cultures were exposed to 20 nm citrate-coated Ag-NPs with a deposition of 30 and 278 ng/cm2 respectively and incubated for 4 h and 24 h. To elucidate whether any effects of Ag-NPs are due to ionic effects, Ag-Nitrate (AgNO3) solutions were aerosolised at the same molecular mass concentrations. RESULTS: Agglomerates of Ag-NPs were detected at 24 h post exposure in vesicular structures inside cells but the cellular integrity was not impaired upon Ag-NP exposures. Minimal cytotoxicity, by measuring the release of lactate dehydrogenase, could only be detected following a higher concentrated AgNO3-solution. A release of pro-inflammatory markers TNF-alpha and IL-8 was neither observed upon Ag-NP and AgNO3 exposures as well as was not affected when cells were pre-stimulated with lipopolysaccharide (LPS). Also, an induction of mRNA expression of TNF-alpha and IL-8, could only be observed for the highest AgNO3 concentration alone or even significantly increased when pre-stimulated with LPS after 4 h. However, this effect disappeared after 24 h. Furthermore, oxidative stress markers (HMOX-1, SOD-1) were expressed after 4 h in a concentration dependent manner following AgNO3 exposures only. CONCLUSIONS: With an experimental setup reflecting physiological exposure conditions in the human lung more realistic, the present study indicates that Ag-NPs do not cause adverse effects and cells were only sensitive to high Ag-ion concentrations. Chronic exposure scenarios however, are needed to reveal further insight into the fate of Ag-NPs after deposition and cell interactions.

Biokinetics of nanoparticles and susceptibility to particulate exposure in a murine model of cystic fibrosis.

BACKGROUND Persons with cystic fibrosis (CF) are at-risk for health effects from ambient air pollution but little is known about the interaction of nanoparticles (NP) with CF lungs. Here we study the distribution of inhaled NP in a murine CF model and aim to reveal mechanisms contributing to adverse effects of inhaled particles in susceptible populations. METHODS Chloride channel defective CftrTgH (neoim) Hgu mice were used to analyze lung function, lung distribution and whole body biokinetics of inhaled NP, and inflammatory responses after intratracheal administration of NP. Distribution of 20-nm titanium dioxide NP in lungs was assessed on ultrathin sections immediately and 24 h after a one-hour NP inhalation. NP biokinetics was deduced from total and regional lung deposition and from whole body translocation of inhaled 30-nm iridium NP within 24 h after aerosol inhalation. Inflammatory responses were assessed within 7 days after carbon NP instillation. RESULTS Cftr mutant females had moderately reduced lung compliance and slightly increased airway resistance compared to wild type mice. We found no genotype dependent differences in total, regional and head deposition or in secondary-organ translocation of inhaled iridium NP. Titanium dioxide inhalation resulted in higher NP uptake by alveolar epithelial cells in Cftr mutants. Instillation of carbon NP induced a comparable acute and transient inflammatory response in both genotypes. The twofold increase of bronchoalveolar lavage (BAL) neutrophils in Cftr mutant compared to wild type mice at day 3 but not at days 1 and 7, indicated an impaired capacity in inflammation resolution in Cftr mutants. Concomitant to the delayed decline of neutrophils, BAL granulocyte-colony stimulating factor was augmented in Cftr mutant mice. Anti-inflammatory 15-hydroxyeicosatetraenoic acid was generally significantly lower in BAL of Cftr mutant than in wild type mice. CONCLUSIONS Despite lacking alterations in lung deposition and biokinetics of inhaled NP, and absence of significant differences in lung function, higher uptake of NP by alveolar epithelial cells and prolonged, acute inflammatory responses to NP exposure indicate a moderately increased susceptibility of lungs to adverse effects of inhaled NP in Cftr mutant mice and provides potential mechanisms for the increased susceptibility of CF patients to air pollution.

Particle Patterning on Lithium Niobate waveguides via photovoltaic tweezers

Successful micro and nano-particle patterning on iron doped lithium niobate waveguides using photovoltaic fields is reported. This technique previously used in bulk crystals is here applied to waveguide configuration. Well defined particle patterns are obtained using two types of planar waveguides (by proton exchanged and swift heavy ion irradiation) and metallic and dielectric neutral particles. The use of waveguide configuration has allowed a reduction of the light exposure time until 3 s, two orders of magnitude smaller than typical values used in bulk.

The intrinsic ability of ribosomes to bind to endoplasmic reticulum membranes is regulated by signal recognition particle and nascent-polypeptide-associated complex.

Signal peptides direct the cotranslational targeting of nascent polypeptides to the endoplasmic reticulum (ER). It is currently believed that the signal recognition particle (SRP) mediates this targeting by first binding to signal peptides and then by directing the ribosome/nascent chain/SRP complex to the SRP receptor at the ER. We show that ribosomes can mediate targeting by directly binding to translocation sites. When purified away from cytosolic factors, including SRP and nascent-polypeptide-associated complex (NAC), in vitro assembled translation intermediates representing ribosome/nascent-chain complexes efficiently bound to microsomal membranes, and their nascent polypeptides could subsequently be efficiently translocated. Because removal of cytosolic factors from the ribosome/nascent-chain complexes also resulted in mistargeting of signalless nascent polypeptides, we previously investigated whether readdition of cytosolic factors, such as NAC and SRP, could restore fidelity to targeting. Without SRP, NAC prevented all nascent-chain-containing ribosomes from binding to the ER membrane. Furthermore, SRP prevented NAC from blocking ribosome-membrane association only when the nascent polypeptide contained a signal. Thus, NAC is a global ribosome-binding prevention factor regulated in activity by signal-peptide-directed SRP binding. A model presents ribosomes as the targeting vectors for delivering nascent polypeptides to translocation sites. In conjunction with signal peptides, SRP and NAC contribute to this specificity of ribosomal function by regulating exposure of a ribosomal membrane attachment site that binds to receptors in the ER membrane.

Occupational exposure to fungi and particles in animal feed industry

Background: Very few studies regarding fungal and particulate matter (PM) exposure in feed industry have been reported, although such contaminants are likely to be a significant contributing factor to several symptoms reported among workers. The purpose of this study has been to characterize fungal and dust exposure in one Portuguese feed industry. Material and Methods: Air and surface samples were collected and subject to further macro- and microscopic observations. In addition we collected other air samples in order to perform real-time quantitative polymerase chain reaction (PCR) amplification of genes from Aspergillus fumigatus and Aspergillus flavus complexes as well as Stachybotrys chartarum. Additionally, two exposure metrics were considered – particle mass concentration (PMC), measured in 5 different sizes (PM0.5, PM1, PM2.5, PM5, PM10), and particle number concentration (PNC) based on results given in 6 different sizes in terms of diameter (0.3 μm, 0.5 μm, 1 μm, 2.5 μm, 5 μm and 10 μm). Results: Species from the Aspergillus fumigatus complex were the most abundant in air (46.6%) and in surfaces, Penicillium genus was the most frequently found (32%). The only DNA was detected from A. fumigatus complex. The most prevalent in dust samples were smaller particles which may reach deep into the respiratory system and trigger not only local effects but also the systemic ones. Conclusions: Future research work must be developed aiming at assessing the real health effects of these co-exposures.

Importance of size-selective particle measuring for assessing occupational exposures: a case study “from field to fork”

Sampling the total air concentration of particulate matter (PM) only provides a basic estimate of exposure that normally not allows correlating with the observed health effects. Therefore is of great importance to recognize the particles size distribution and, particularly, the exposure to fine particles (≤ 2.5 μm). This particles dimension corresponds to the respirable fraction, the one that can implicate local and systemic effects due to particle deposition and clearance from the lungs and transport within the organism. This study intended to describe occupational exposure to PM2.5 in three units related with swine production and consumption, namely: feed production, swine production and swine slaughterhouse. A size-selective particle measuring in five to six workplaces of each unit was performed. Measurements of PM were done using a portable direct-reading hand-held equipment (Lighthouse, model 3016 IAQ). Data showed slaughterhouse unit with higher values, with values ranging from 0.030 to 0.142 mg/m3 (0.073 + 0.043), being the cutting room the workplace with higher values. In feed production unit, values were between 0.026 and 0.033 mg/m3 (0.028 + 0.003) with the warehouse of pharmacy products as the workplace with higher values. Finally, in swine unit values ranged from 0.006 to 0.048 mg/m3 (0.023 + 0.017) with the batteries area presenting the higher values. PM can be rich in fungi and bacteria and their metabolites, such as endotoxins and mycotoxins. Previous publications already showed high contamination in these occupational settings and particles can have an important role in exposure since can easily act as carrier of these agents. Data acquired allow not only a better prediction of particle penetration into respiratory regions of the respiratory tract, but also a better estimation of PM health effects. Moreover, data permit to identify the workplaces where investment should be made to prevent and reduce exposure.

Occupational exposure to particles during hotel´s rooms cleaning

Background: The majority of studies investigated ambient particles, although in most industrialized countries people spend most of their time indoors and significant emissions of fine and ultrafine particles leading to human exposure are caused by various indoor tasks, including cleaning tasks. Objective: To characterize the occupational exposure to particles during cleaning of hotel's rooms. Methodology: Measurements of mass concentration and particle number concentration were performed before and during cleaning tasks in two rooms with different floor types (wood and carpet) with the equipment Lighthouse, model 3016 IAQ. Results: Considering mass concentration, particles with higher were responsable for higher leves of contamination, particularly PM5.0 and PM10.0. However, considering the particle number concentration, the smaller particle size obtained the higher values. Conclusion: It was observed higher number of particles of the smaller size in all tasks, which is associated with worse health effects. It was observed that the room with wood in the floor has lower values when compared to the room with carpet. The tasks with greater exposure were the 'vacuuming' and 'clean up powder'.

Importance of size-selective particle sampling for assessing occupational exposures: results from three different occupational settings

Sampling the total air concentration of particulate matter (PM) only provides a basic estimate of exposure that normally not allows correlating with the observed health effects. Therefore is of extreme importance to know the particles size distribution and, in more detail, the exposure to fine particles (≤ 2.5 µm). This particles dimension corresponds to the respirable fraction. This particle fraction can result, besides local effects, in systemic effects due to particle deposition and clearance from the lungs and transport within the organism. This study intended to describe occupational exposure to PM2.5 in three different units located near Lisbon and related with occupational exposure to organic dust, namely: swine and poultry feed production and waste management.

Children exposure to indoor ultrafine particles in urban and rural school environments

Extended exposure to ultrafine particles (UFPs) may lead to consequences in children due to their increased susceptibility when compared to older individuals. Since children spend in average 8 h/day in primary schools, assessing the number concentrations of UFPs in these institutions is important in order to evaluate the health risk for children in primary schools caused by indoor air pollution. Thus, the purpose of this study was to assess and determine the sources of indoor UFP number concentrations in urban and rural Portuguese primary schools. Indoor and outdoor ultrafine particle (UFP) number concentrations were measured in six urban schools (US) and two rural schools (RS) located in the north of Portugal, during the heating season. The mean number concentrations of indoor UFPs were significantly higher in urban schools than in rural ones (10.4 × 10(3) and 5.7 × 10(3) pt/cm(3), respectively). Higher UFP levels were associated with higher squared meters per student, floor levels closer to the ground, chalk boards, furniture or floor covering materials made of wood and windows with double-glazing. Indoor number concentrations of ultrafine-particles were inversely correlated with indoor CO2 levels. In the present work, indoor and outdoor concentrations of UFPs in public primary schools located in urban and rural areas were assessed, and the main sources were identified for each environment. The results not only showed that UFP pollution is present in augmented concentrations in US when compared to RS but also revealed some classroom/school characteristics that influence the concentrations of UFPs in primary schools.

Exposure of children to ultrafine particles in primary schools in Portugal

Children spend a large part of their time at schools, which might be reflected as chronic exposure. Ultrafine particles (UFP) are generally associated with a more severe toxicity compared to fine and coarse particles, due to their ability to penetrate cell membranes. In addition, children tend to be more susceptible to UFP-mediated toxicity compared to adults, due to various factors including undeveloped immune and respiratory systems and inhalation rates. Thus, the purpose of this study was to determine indoor UFP number concentrations in Portuguese primary schools. Ultrafine particles were sampled between January and March 2014 in 10 public primary schools (35 classrooms) located in Porto, Portugal. Overall, the average indoor UFP number concentrations were not significantly different from outdoor concentrations (8.69 × 10(3) vs. 9.25 × 10(3) pt/cm(3), respectively; considering 6.5 h of indoor occupancy). Classrooms with distinct characteristics showed different trends of indoor UFP concentrations. The levels of carbon dioxide were negatively correlated with indoor UFP concentrations. Occupational density was significantly and positively correlated with UFP concentrations. Although the obtained results need to be interpreted with caution since there are no guidelines for UFP levels, special attention needs to be given to source control strategies in order to reduce major particle emissions and ensure good indoor air quality.