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 microm). 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 microm) 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.

Electron energy loss spectroscopy for analysis of inhaled ultrafine particles in rat lungs.

Epidemiologic studies have associated cardiovascular morbidity and mortality with ambient particulate air pollution. Particles smaller than 100 nm in diameter (ultrafine particles) are present in the urban atmosphere in very high numbers yet at very low mass concentration. Organs beyond the lungs are considered as targets for inhaled ultrafine particles, whereby the route of particle translocation deeper into the lungs is unclear. Five rats were exposed to aerosols of ultrafine titanium dioxide particles of a count median diameter of 22 nm (geometric standard deviation, GSD 1.7) for 1 hour. The lungs were fixed by intravascular perfusion of fixatives immediately thereafter. TiO(2) particles in probes of the aerosol as well as in systematic tissue samples were analyzed with a LEO 912 transmission electron microscope equipped with an energy filter for elemental microanalysis. The characteristic energy loss spectra were obtained by fast spectrum acquisition. Aerosol particles as well as those in the lung tissue were unambiguously identified by electron energy loss spectroscopy. Particles were mainly found as small clusters with a rounded shape. Seven percent of the particles in the lung tissue had a needle-like shape. The size distribution of the cluster profiles in the tissue had a count median diameter of 29 nm (GSD 1.7), which indicates no severe clustering or reshaping of the originally inhaled particles. Electron energy loss spectroscopy and related analytical methods were found to be suitable to identify and localize ultrafine titanium dioxide particles within chemically fixed and resin-embedded lung tissue.

Influence of airspace geometry and surfactant on the retention of man-made vitreous fibers (MMVF 10a).

Inhaled and deposited man-made vitreous fibers (MMVF) 10a (low-fluorine preparation of Schuller 901 insulation glass) were studied by electron microscopy in hamster lungs, fixed by intravascular perfusion within 23 +/- 2 min (SD) of the initial inhalation. We found fibers on the surfaces of conducting airways and alveoli. In the airways, 89% of the fibers were totally and 11% partially covered by lining-layer material. In the alveoli, 32% of the fibers were totally submersed; others touched the alveolar wall, stuck at one end, bridging the airspace. Studies in a surface balance showed that fibers were immersed into the aqueous subphase by approximately 50% at film surface tensions of 20-25 mJ/m2) and were submersed (totally immersed; i.e., totally surrounded by fluid) at approximately 10 mJ/m2). Fibers were also found to be phagocytosed by macrophages. We found a substantial number of particle profiles within alveolar blood capillaries. Fiber length and alveolar geometry appear to be important limiting factors for the submersion of vitreous fibers into the lungs' surface lining layer.