Combustion-derived ultrafine particles transport organic toxicants to target respiratory cells

Abstract

Epidemiologic evidence supports associations between inhalation of fine and ultrafine ambient particulate matter [aerodynamic diameter < or = 2.5 microm (PM2.5)] and increases in cardiovascular/respiratory morbidity and mortality. Less attention has been paid to how the physical and chemical characteristics of these particles may influence their interactions with target cells. Butadiene soot (BDS), produced during combustion of the high-volume petrochemical 1,3-butadiene, is rich in polynuclear aromatic hydrocarbons (PAHs), including known carcinogens. We conducted experiments to characterize BDS with respect to particle size distribution, assembly, PAH composition, elemental content, and interaction with respiratory epithelial cells. Freshly generated, intact BDS is primarily (> 90%) PAH-rich, metals-poor (nickel, chromium, and vanadium concentrations all < 1 ppm) PM2.5, composed of uniformly sized, solid spheres (30-50 nm) in aggregated form. Cells of a human bronchial epithelial cell line (BEAS-2B) exhibit sequential fluorescent responses--a relatively rapid (approximately 30 min), bright but diffuse fluorescence followed by the slower (2-4 hr) appearance of punctate cytoplasmic fluorescence--after BDS is added to medium overlying the cells. The fluorescence is associated with PAH localization in the cells. The ultrafine BDS particles move down through the medium to the cell membrane. Fluorescent PAHs are transferred from the particle surface to the cell membrane, cross the membrane into the cytosol, and appear to accumulate in lipid vesicles. There is no evidence that BDS particles pass into the cells. The results demonstrate that uptake of airborne ultrafine particles by target cells is not necessary for transfer of toxicants from the particles to the cells.

Figure 1

(A) An SEM image illustrating the lacy openwork character typical of the BDS aggregates; individual, solid, spherical particles, 50–70 nm in diameter, are the fundamental structural units of the aggregates. (B) A TEM image of BDS showing individual spheres, 30–50 nm in diameter, arranged in branching clusters. The difference in diameter of the spheres in the SEM versus TEM images results from the 10–20 nm gold/palladium conductive coating that was applied to the SEM samples. (C) A TEM image of a portion of the surface of a BEAS-2B cell with individual spherical particles, 30–50 nm in diameter, and small aggregates (arrows) immediately adjacent to the cell membrane. Cells were photographed after 42 hr exposure.

Figure 2

Fluorescence localized in punctate cytoplasmic vesicles of BEAS-2B cells. Cells were photographed 4 hr after BDS, without carrier, was sprinkled onto the surface of the BEGM overlying the cells. Excitation/emission wavelengths = 360/420 nm. Magnification, 400×.

Figure 3

GC/MS ion chromatograms of (A) BDS extract (total ion), (B) cells exposed to BDS sprinkled on medium surface (selected ion monitoring (SIM)], (C) BDS added to a Transwell placed over cells (SIM), and (D) BDS added to a Transwell placed over an ODS disk (SIM). The same 202 m/z cluster was observed in all cases. Interference with monitoring of other PAH masses was due to cell or method contaminants, as noted. The GC/MS analyses were conducted as described in “Materials and Methods,” except that monitoring was in the selective ion mode.