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The impact of nanoparticles (NPs) in medicine and biology has increased rapidly in recent years. Gold NPs have advantageous properties such as chemical stability, high electron density and affinity to biomolecules, making them very promising candidates as drug carriers and diagnostic tools. However, diverse studies on the toxicity of gold NPs have reported contradictory results. To address this issue, a triple cell co-culture model simulating the alveolar lung epithelium was used and exposed at the air-liquid interface. The cell cultures were exposed to characterized aerosols with 15 nm gold particles (61 ng Au/cm2 and 561 ng Au/cm2 deposition) and incubated for 4 h and 24 h. Experiments were repeated six times. The mRNA induction of pro-inflammatory (TNFalpha, IL-8, iNOS) and oxidative stress markers (HO-1, SOD2) was measured, as well as protein induction of pro- and anti-inflammatory cytokines (IL-1, IL-2, IL-4, IL-6, IL-8, IL-10, GM-CSF, TNFalpha, INFgamma). A pre-stimulation with lipopolysaccharide (LPS) was performed to further study the effects of particles under inflammatory conditions. Particle deposition and particle uptake by cells were analyzed by transmission electron microscopy and design-based stereology. A homogeneous deposition was revealed, and particles were found to enter all cell types. No mRNA induction due to particles was observed for all markers. The cell culture system was sensitive to LPS but gold particles did not cause any synergistic or suppressive effects. With this experimental setup, reflecting the physiological conditions more precisely, no adverse effects from gold NPs were observed. However, chronic studies under in vivo conditions are needed to entirely exclude adverse effects.

Sixteen beagle dogs were housed in four large chambers under minimum restraint. They were exposed for 16 months to clean air and individual baseline data of markers were obtained. For 13 months, eight dogs were further exposed to clean air and eight dogs for 6 h/d to 1-microm MMAD (mass median aerodynamic diameter) acidic sulfate particles carrying 25 micromol H(+) m(-3) into their lungs. To establish functional responses (lung function, cell and tissue integrity, redox balance, and non-specific respiratory defense capacity), each exposed animal served as its own control. To establish structural responses, the eight non-exposed animals served as controls. Acidic particles were produced by nebulization of aqueous sodium hydrogen sulfate at pH 1.5. Only subtle exposure-related changes of lung function and structure were detected. A significant increase in respiratory burst function of alveolar macrophages points to a marginal inflammatory response. This can be explained by the significant production of prostaglandin E(2), activating cyclooxygenase-dependent mechanisms in epithelia and thus inhibiting lung inflammation. The non-specific defense capacity was slightly affected, giving increased tracheal mucus velocity and reduced in vivo dissolution of moderately soluble test particles. Hypertrophy and hyperplasia of bronchial epithelia were not observed, but there was an increase in volume density of bronchial glands and a shift from neutral to acidic staining of epithelial secretory cells in distal airways. The acidic exposure had thus no pathophysiological consequences. It is therefore unlikely that long-term inhalation of acidic particles is associated with a health risk.

Both epidemiological and toxicological studies indicate that inhalation and subsequent deposition of airborne particles into the lungs have adverse health effects. Recently, the ultrafine particle (UfP) fraction (diameter < 100 nm) has received particular attention, as their small size may lead to more toxic properties. In this study we summarize the current knowledge on the dosimetry of inhaled particles (including UfPs) with a focus on recent data on translocation of UfPs into secondary target organs (such as brain and heart) suggesting that the lifetime dose of ambient UfPs in secondary target organs is about 10(11) particles. Furthermore, we highlight the main pathways of particle induced toxicity and the reasons for the potentially higher toxicity of UfPs. Finally, we discuss recent evidence indicating that (BET) surface area is the single most relevant dose metric for the toxicity of UfPs, which has important implications for regulatory measures on the toxicity of ambient and engineered particles.

The geometry of commercially available perfusion chambers designed for harbouring three membrane-based cell cultures was modified for reliable and dose-controlled air-liquid interface (ALI) exposures. Confluent A549 epithelial cells grown on membranes were integrated in the chamber system and supplied with medium from the chamber bottom. Cell viability was not impaired by the conditions of ALI exposure without particles. Expression of the inflammatory cytokines interleukin 6 and interleukin 8 by A549 cells during ALI exposure to filtered air for 6h and subsequent stimulation with tumor necrosis factor was not altered compared to submersed controls, indicating that the cells maintained their functional integrity. Ultrafine carbonaceous model particles with a count median mobility diameter of about 95+/-5 nm were produced by spark discharge at a stable concentration of about 2 x 10(6) cm(-3) and continuously monitored for accurate determination of the exposure dose. Delivery to the ALI exposure system yielded a homogeneous particle deposition over the membranes with a deposition efficiency of 2%. Mid dose exposure of A549 cells to this aerosol for 6h yielded a total particle deposition of (2.6+/-0.4) x 10(8) cm(-2) corresponding to (87+/-23) ng cm(-2). The 2.7-fold (p < or = 0.05) increased transcription of heme oxygenase-1 indicated a sensitive antioxidant and stress response, while cell viability did not reveal a toxic mechanism.

Recently concern has been raised about health effects related to environmental sulfur and/or acidic aerosols. To assess long-term effects on respiratory lung function, 8 beagle dogs were exposed over a period of 13 mo for 16.5 h/day to 1.0 microm neutral sulfite aerosol with a particle associated sulfur(IV) concentration of 0.32 mg m(-3) and for 6 h/day to 1.1 microm acidic sulfate aerosol providing an hydrogen ion concentration of 15.2 micromol m(-3) for inhalation. Prior to exposure the dogs were kept under clean air conditions for 16 mo to establish physiological baseline values for each dog. A second group of eight dogs (control) was kept for the entire study under clean air conditions. Nonspecific defense mechanisms in the airways and in the peripheral lung were studied during chronic exposure of the combination of neutral sulfur(IV) and acidic sulfur(VI) aerosols. No functional changes of tracheal mucus velocity were found, in agreement with unchanged morphometry of the airways. However, the exposure resulted in changes of several alveolar macrophage (AM) mediated particle clearance mechanisms: (1) Based on in vivo clearance analysis and cultured AM studies using moderately soluble cobalt oxide particles, intracellular particle dissolution was significantly reduced since phagolysosomal proton concentration was decreased. We deduce exposure-related malfunction of proton pumps bound to the phagolysosomal membrane as a result of an increase of cytosolic proton concentration. (2) Based on in vivo clearance analysis using insoluble polystyrene particles, AM-mediated particle transport from the lung periphery toward ciliated terminal bronchioli and further to the larynx was significantly reduced. Activation of epithelial type II cells at the entrance of alveoli was inferred from observed type II cell proliferation at those alveolar ridges and enhanced secretion of alkaline phosphatase in the fluid of bronchoalveolar lavages. As a result, hypersecretion of chemotactic mediators by activated type II cells at these loci led to the observed decrease of particle transport toward ciliated bronchioli. (3) Based on in vivo clearance analysis using insoluble polystyrene particles, particle transport from the alveolar epithelium into interstitial tissues was increased and (4) particle transport to the tracheobronchial lymph nodes was significantly enhanced. Particle transport into interstitial tissues is the most prominent clearance pathway from the canine alveolar epithelium. We conclude that the deteriorated particle transport toward ciliated terminal bronchioli resulted in an enhanced particle transport across the epithelial membrane into interstitial tissues and the lymphatic drainage. The observed alterations in alveolar macrophage-mediated clearance mechanisms during chronic exposure of these air pollutants indicate an increased risk of health.

The motivation of simulating real-world environmental exposure in a number of long-term studies with dogs was to address the question of whether or not perpetual inhalation of air pollutants can initiate diseases in healthy lungs and can thus contribute to the increasing prevalence of respiratory diseases in industrialized countries. The major conclusion of this article is that this question has to be answered in the negative for the simultaneous inhalation of the major constituents of combustion-related air pollution, particle-associated sulfur(IV), and particle-associated hydrogen ions. Over 13 mo, 8 healthy beagle dogs were exposed in 2 whole-body chambers daily for 16.5 h to 1 microm neutral sulfite [sulfur(IV)] particles at a mass concentration of 1.5 mg m-3 and for 6 h to 1.1 microm acidic sulfate particles carrying 15 micromol m-3 hydrogen ions into the canine lungs. This longitudinal study was characterized by repeated observations of individual respiratory response patterns. To establish baseline data the dogs were repeatedly examined preexposure while the chambers were ventilated over 16 mo with clean air. Each individual served thus as its own control. Another eight dogs served as additional controls. They were housed in 2 chambers ventilated with clean air over the entire study period of 29 mo. To assess response patterns, respiratory lung function tests were performed pre- and postexposure, segmental lung lavages were repeatedly performed to obtain epithelial lining fluid from the lungs for analysis of cell content, cell function, and biochemical indicators of lung injury, and radiolabeled test particles were used to study pathways of intrapulmonary particle elimination. At the end of the study, the lungs of all animals were morphologically and morphometrically examined. Functional and structural responses were finally compared to those observed previously as a result of a sole exposure of canine lungs to neutral sulfite particles over 10 mo (Heyder et al., 1992). Interactions between responses induced by neutral sulfite and acidic sulfate particles occurred, but antagonism rather than synergism was observed. The responses induced by sulfur(IV) were less pronounced, not detectable, or even reversed when hydrogen ions were also delivered to the lungs. On the other hand, responses not induced by the sole exposure to sulfur(IV) were observed: The activity of alkaline phosphatase was elevated and type II pneumocytes proliferated. It can, however, be concluded that long-term exposure of healthy lungs to particle-associated neutral sulfur(IV) and hydrogen ions at concentration close to ambient levels causes subtle respiratory responses but does not initiate pathological processes in the lungs. In other words, the perpetual inhalation of sulfur(IV) and hydrogen ions from the atmospheric environment presents no health risk to the healthy lungs. It is thus also very unlikely that respiratory diseases can be initiated by the inhalation of these pollutants.

Lung clearance of a well-defined uniform and respirable material was conducted to aid in the development of models used to relate inhalation of inorganic hazardous particles to organ doses and bioassay measurements, and in particular to aid in the extrapolation of animal data to humans. In the present study, lung clearance was investigated in Long-Evans rats using monodisperse, porous, 0.8- and 1.7-microns-diameter cobalt oxide (Co3O4) test particles. An advanced inhalation technique for rats using endotracheal intubation yielded exclusive particle deposition in the pulmonary region without external pelt contamination, thus allowing for clearance studies starting directly after inhalation. The kinetics of lung clearance was distinguished between the two dominant clearance mechanisms of mechanical particle transport to the larynx and translocation of dissolved particle material to blood. A particle fraction of about 40% was cleared by short-term particle transport to the larynx, both the long-term particle transport rate and the translocation rate of dissolved particle material given as fractional rates of the retained particle mass in the lungs were not constant with time. The former declined from 0.03 to 0.004 d-1 during 6 months after inhalation. The latter depended on the specific surface area of the porous particles and increased with time from 0.08 and 0.04 d-1 for 0.8- and 1.7-microns particles, respectively. The results obtained were compared to previously reported data obtained from Fischer-344 rats and HMT rats. These were part of a previously reported interspecies comparison of lung clearance followed in seven species, including humans, and using the same batches of Co3O4 test particles. Long-term lung retention was similar in Long-Evans rats and HMT rats but decreased faster for both particle sizes than in Fischer-344 rats, as a result of a significantly faster translocation of dissolved material from the test particles to blood. Mechanical particle transport to the larynx was comparable in all three species.

Lung retention of 57Co in dogs after the inhalation of physically and chemically uniform particles of Co compounds was similar, indicating little biological variability. The retention of Co oxide particles ranging from 0.3 micron to 2.7 micron geometric diameter, however, depended markedly on their physicochemical parameters. Measuring the retention by a gamma camera, and analyzing excreta and blood samples enabled the distinction of different clearance pathways from the lungs particularly with the use of results of some metabolic studies. Particle dissolution was the predominant clearance pathway. Particle dissolution half-times ranged from 6 to 80 d proportional to the size of the particles. Organ analysis yielded information on the fate of the long-term burden of Co in the lungs and other organs. A fraction less than 10% of the initial lung burden was retained in the lungs of all dogs with a biological half-time of 400 d presumably after being transformed into a nonparticulate state. There were cellular structures in the tracheobronchial tree which accumulated Co significantly. These studies on dogs suggest the dose after human exposure to well-defined Co aerosols can be accurately estimated. Whereas risk assessment after the exposure to undefined aerosols containing radionuclides of Co will mostly be impossible because retention varies widely with the varying physicochemical properties of the aerosol particles.

Investigations on the physical factors influencing the efficiency of 3 kinds of nebulizers (Wiesbadener Doppelinhalator, Heyer jet nebulizer, and Monaghan ultrasonic nebulizer) used to administer pharmaceutical agents are described. The airflow, the waterflow, and the concentration of the nebulized pharmaceutical agents in the air were determined. It was found that, for the jet nebulizers, the concentration of nebulized pharmaceuticals decreased considerably during the vaporization. The particle size distribution was measured with spiral centrifuge. The mass median diameter of the unevaporated aerosols was 3.4 mum for the Wiesbadener, 7.4 mum for the Heyer, and 5.2 mum for the Monaghan. The deposition of an aerosol produced with the Wiesbadener was measured in a glass model of the upper airways (mouth, trachea, and bifurcation). An estimate of the fraction of the aerosol that can pass the bifurcation was made. A correction for the change in the aerosol particle diameter due to the high humidity in the human lung was introduced. It was found that 35 per cent of the mass of the aerosol produced with this nebulizer can pass the human bifurcation (tidal volume, 500 ml; inhalation frequency, 12 per min).