J. Heyder

Ultrafine Particles Cross Cellular Membranes by Nonphagocytic Mechanisms in Lungs and in Cultured Cells

High concentrations of airborne particles have been associated with increased pulmonary and cardiovascular mortality, with indications of a specific toxicologic role for ultrafine particles (UFPs; particles < 0.1 μm). Within hours after the respiratory system is exposed to UFPs, the UFPs may appear in many compartments of the body, including the liver, heart, and nervous system. To date, the mechanisms by which UFPs penetrate boundary membranes and the distribution of UFPs within tissue compartments of their primary and secondary target organs are largely unknown. We combined different experimental approaches to study the distribution of UFPs in lungs and their uptake by cells. In the in vivo experiments, rats inhaled an ultrafine titanium dioxide aerosol of 22 nm count median diameter. The intrapulmonary distribution of particles was analyzed 1 hr or 24 hr after the end of exposure, using energy-filtering transmission electron microscopy for elemental microanalysis of individual particles. In an in vitro study, we exposed pulmonary macrophages and red blood cells to fluorescent polystyrene microspheres (1, 0.2, and 0.078 μm) and assessed particle uptake by confocal laser scanning microscopy. Inhaled ultrafine titanium dioxide particles were found on the luminal side of airways and alveoli, in all major lung tissue compartments and cells, and within capillaries. Particle uptake in vitro into cells did not occur by any of the expected endocytic processes, but rather by diffusion or adhesive interactions. Particles within cells are not membrane bound and hence have direct access to intracellular proteins, organelles, and DNA, which may greatly enhance their toxic potential.

Deposition of particles in the human respiratory tract in the size range 0.005–15 μm

Abstract Experimentally determined total and regional deposition data are presented for breathing monodisperse aerosols of a wide particle size range at different patterns through the mouth and nose. From these data simple analytical expressions were derived for the efficiencies of the nasal passages, larynx, upper and lower ciliated thoracic airways and the nonciliated portion of the lungs in collecting particles from inspired aerosols. Thus, empirical expressions are now available for the calculation of total and regional deposition in the human respiratory tract for particles of any size and density inspired at any pattern through the mouth or nose.

Distribution Pattern of Inhaled Ultrafine Gold Particles in the Rat Lung

The role of alveolar macrophages in the fate of ultrafine particles in the lung was investigated. Male Wistar-Kyoto rats were exposed to ultrafine gold particles, generated by a spark generator, for 6 h at a concentration of 88 μg/m3 (4 × 106/cm3, 16 nm modal mobility diameter). Up to 7 days, the animals were serially sacrificed, and lavaged cells and lung tissues were examined by transmission electron microscopy. The gold concentration/content in the lung, lavage fluid, and blood was estimated by inductively coupled plasma–mass spectrometry. Gold particles used were spherical and electron dense with diameters of 5–8 nm. The particles were individual or slightly agglomerated. By inductively coupled plasma–mass spectrometry analysis of the lung, 1945 ± 57 ng (mean ± SD) and 1512 ± 184 ng of gold were detected on day 0 and on day 7, respectively, indicating that a large portion of the deposited gold particles was retained in the lung tissue. In the lavage fluid, 573 ± 67 ng and 96 ± 29 ng were found on day 0 and day 7, respectively, which means that 29% and 6% of the retained gold particles were lavageable on these days. A low but significant increase of gold (0.03 to 0.06% of lung concentration) was found in the blood. Small vesicles containing gold particles were found in the cytoplasm of alveolar macrophages. In the alveolar septum, the gold particles were enclosed in vesicles observed in the cytoplasm of alveolar type I epithelial cells. These results indicate that inhaled ultrafine gold particles in alveolar macrophages and type I epithelial cells are processed by endocytotic pathways, though the uptake of the gold particles by alveolar macrophages is limited. To a low degree, systemic particle translocation took place.

Generation of ultrafine particles by spark discharging

Ultrafine carbon, metal, and metal oxide particles were generated with a commercially available spark generator designed for the production of carbon particles. Aerosols with number concentrations up to 107 cm−3 were produced at flow rates up to 150 lpm. Lognormal size distributions with modal diameters in the range of 18–150 nm and geometric standard deviations of about 1.5 were obtained. The chemical composition, size, number concentration, morphology, and surface area of the particles were varied, and the generation of particles with fixed characteristics could be maintained over many hours. The particle characteristics, however, could not be varied independently. For a certain chemical composition only size and number concentration were variable; morphology and surface area were fixed regardless of particle size. The particles grow by coagulation of primary particles formed by nucleation. The coagulated particles can either stick together and maintain their identity or fuse together and lose their identity. Each material used for the generation of ultrafine particles is thus associated with a certain morphology and surface area: silver with a low mass-related BET surface area (20 m2 g−1), metal oxides and iridium with a low-to-intermediate BET surface area (50 m2 g−1 for cadmium oxide, 120 m2 g−1 for iridium, and 300 m2 g− 1 for ferric oxide), and carbon with a large BET surface area (750 m2 g−1). Iridium, on the other hand, has a huge volume-related BET surface area (2800 m2 cm−3). It was not possible to generate ultrafine carbon particles without contaminations with the generator. However, these contaminations could be decreased in this study from 25% to 6% by replacing organic components of the generator by pure inorganic components.

Fate and toxic effects of inhaled ultrafine cadmium oxide particles in the rat lung.

Female Fischer 344 rats were exposed to ultrafine cadmium oxide particles, generated by spark discharging, for 6 h at a concentration of 70 μg Cd/m3 (1× 106/cm3) (40 nm modal diameter). Lung morphology and quantification of Cd content/concentration by inductively coupled plasma (ICP)–mass spectrometry were performed on days 0, 1, 4, and 7 after exposure. Cd content in the lung on day 0 was 0.53± 0.12 μg/lung, corresponding to 19% of the estimated total inhaled cumulative dose, and the amount remained constant throughout the study. In the liver no significant increase of Cd content was found up to 4 days. A slight but statistically significant increase was observed in the liver on day 7. We found neither exposure-related morphological changes of lungs nor inflammatory responses in lavaged cells. Another group of rats were exposed to a higher concentration of ultrafine CdO particles (550 μg Cd/m3 for 6 h, 51 nm modal diameter). The rats were sacrificed immediately and 1 day after exposure. The lavage study performed on day 0 showed an increase in the percentage of neutrophils. Multifocal alveolar inflammation was seen histologically on day 0 and day 1. Although the Cd content in the lung was comparable between day 0 and day 1 (3.9 μg/lung), significant elevation of Cd levels in the liver and kidneys was observed on both days. Two of 4 rats examined on day 0 showed elevation of blood cadmium, indicating systemic translocation of a fraction of deposited Cd from the lung in this group. These results and comparison with reported data using fine CdO particles indicate that inhalation of ultrafine CdO particles results in efficient deposition in the rat lung. With regard to the deposition dose, adverse health effects of ultrafine CdO and fine CdO appear to be comparable. Apparent systemic translocation of Cd took place only in animals exposed to a high concentration that induced lung injury.

Cardiovascular responses in unrestrained WKY rats to inhaled ultrafine carbon particles

Based on epidemiologic observations, the issue of adverse health effects of inhaled ultrafine particles (UFP) is currently under intensive discussion. We therefore examined cardiovascular effects of UFP in a controlled animal exposure on young, healthy WKY rats. Short-term exposure (24 h) to carbon UFPs (38 nm, 180 μg m−3), generated by spark discharging, induced a mild but consistent increase in heart rate (18 bpm, 4.8%), which was associated with a significant decrease in heart-rate variability during particle inhalation. The timing and the transient character of these responses point to a particle induced alteration of cardiac autonomic balance, mediated by a pulmonary receptor activation. After 24 h of inhalation exposure, bronchoalveolar lavage revealed significant but low-grade pulmonary inflammation (clean air 1.9% vs. UFPs 6.9% polymorphonuclear cells) and on histopathology sporadic accumulation of particle-laden macrophages was found in the alveolar region. There was no evidence of an inflammation-mediated increase in blood coagulability, as UFP inhalation did not induce any significant changes in plasma fibrinogen or factor VIIa levels and there were no prothrombotic changes in the lung or the heart at both the protein and mRNA level. Histological analysis revealed no signs of cardiac inflammation or cardiomyopathy. This study therefore provides toxicological evidence for UFP-associated pulmonary and cardiac effects in healthy rats. Our findings suggest that the observed changes are mediated by an altered sympatho-vagal balance in response to UFP inhalation, but do not support the concept of an inflammation-mediated prothrombotic state by UFP.

Generation and properties of a condensation aerosol of di-2-ethylhexyl sebacate (DES)—I: Description of the generator

Abstract In an improved generator of the Sinclair-La Mer type monodisperse droplets of di-2-ethylhexyl sebacate (DEHS) in the 0·08–4 ,μm dia. range were produced by condensation of DEHS-vapour on sodium chloride nuclei in a stream of pure nitrogen. The nuclei were produced by electrical heating of a NaCl-coated platinum wire. Their size and concentration depend on the nitrogen flow rate and the heating current as proved by electron microscopy and monitoring of the nuclei concentration. The final size distributions of the DEHS aerosols were evaluated by optical spectrometry. The geometric standard deviation of the size distributions varied between 1·035 and 1·06.

A laser spectrometer for size analysis of small airborne particles

Abstract The particle size distribution in an aerosol is measured by analysing the light scattered by each particle in a nearly forward direction. The particles are illuminated with a laser beam, the light scattered is collected by a microscope objective and passed to a red sensitive photomultiplier, the amplified output pulses of which are stored in a multichannel pulse height analyser. The signal-to-noise ratio of the spectrometer makes it possible to measure particles down to 0–1 μm in diameter. This sensitivity was obtained by focusing both the laser beam and the aerosol stream.

Intercomparison of regional deposition of aerosol particles in the human respiratory tract and their long-term elimination

Human chest clearance of Teflon particles with an aerodynamic diameter of 4.7 micrometers tagged with 198Au or 111In was studied with two apparatuses and two gamma-ray spectrometers for the external detection of the activity deposited in the respiratory tract. Approximately the same chest retention function was measured with two gamma-ray spectrometers when the subjects inhaled equal aerosols under equal breathing conditions. The long-term clearance rate following the short-term elimination of particles from ciliated airways was slower for Teflon particles (mean half-time 105 days for 111In-labeled particles and 128 days for 198Au-labeled particles) than for iron oxide particles of the same size (mean half-time about 60 days). It is suggested that insoluble particles of this size studied are cleared with a half-time of about 120 days within the first 2 weeks after completion of mucociliary clearance. Regional deposition did not differ between the iron oxide and Teflon particles.

Interaction of Diffusional and Gravitational Particle Transport in Aerosols

The interaction of diffusional and gravitational particle transport was determined by studying with optical instruments concentrations during passage of monodisperse polystyrene and sebacate aerosols through filters. Particle losses in the filters due to simultaneous diffusional and gravitational particle transport onto filter surfaces can be described by Although deposition due to pure diffusional particle transport dD or pure gravitational particle transport dG exhibits a different time dependence in filters differing from one another as much as cylindrical tubes, granular beds of spherical glass beads, and human lungs, and although the particle size of minimum deposition varies among these filters, this empirical equation holds for particle deposition in all of these filters. This equation can thus account for simultaneous diffusional and gravitational particle transport on any surface.