(167c) Hydrocarbons Adsorbed to Combustion-Derived PM Localize to Lipid Droplets and Activate Oxidative Stress Response Genes in Respiratory Cells

Airborne particulates are of increasing concern, not only for their contribution to ambient pollution, but also for the toxic health effects they elicit. The health effects have been attributed both to the particles themselves, especially in the readily inhalable fine (<2.5 µm) and ultrafine (<0.1 µm) size ranges and to inorganic and organic chemicals associated with the particles. Included among the organic chemicals are polynuclear aromatic hydrocarbons (PAHs) that are adsorbed to the surface of the particles. Incomplete combustion of organic substrates leads to the generation of complex airborne particulates e.g. those found in cigarette smoke, automobile (gasoline or diesel) exhaust, and petrochemical flares.

Diesel exhaust and cigarette smoke are among the most frequently studied ?real-world' examples of complex combustion-derived particulate mixtures. In both cases, there is a growing body of literature that emphasizes the distinction between the toxicity of the particles versus the toxicity of chemicals adsorbed to the particles. Another source of complex particulate environmental contamination is flaring of fugitive volatile compounds by industry. In these settings, volatiles that escape the processing stream or that remain unused are combusted, as strict regulations are in place limiting the amounts of highly reactive volatile organic compounds, such as 1,3-butadiene (BD), that can be released to the atmosphere.

BD is a high-volume, aliphatic hydrocarbon byproduct of petroleum refining and is used in the manufacture of synthetic rubber and other elastomers. The United States' capacity for BD production has been estimated to be ~3 billion lbs/year, with many of the producers being located in Texas and Louisiana. Butadiene soot (BDS), generated during the combustion of BD, is both a model mixture and a real-life example of a petrochemical product of incomplete combustion with the potential both for environmental contamination and for contributing to health problems.

Free BDS particles have been found apposed to the luminal surface of lung epithelium in mice exposed to BDS by inhalation.

BDS is a metals-poor, organic-rich mixture of ultrafine (30-50 nm) carbonaceous particles to which hundreds of PAH species (100-400 amu) are adsorbed (1,2). Sixteen percent of the total weight of fresh BDS is comprised of PAHs, including benzo(a)pyrene [B(a)P] and other carcinogens, many of which display a characteristic blue or blue-green fluorescence in organic solutions.

Human bronchoepithelial cells exposed to BDS develop blue fluorescence, which over time becomes localized in discrete cytoplasmic vesicles (2). Following BDS exposure, these cells display the same profile of extractable PAHs as the parent BDS. The fluorescence does not develop if the cells are exposed to carbon black instead of BDS, or if the BDS is extracted with organic solvents before the soot particles are presented to the cells.

Lipid droplets are spherical organelles ranging in diameter from 50 nm at formation up to 200 µm in mature adipocytes, with the majority being ~1 µm in mammalian cells. Initially, lipid droplets were regarded as repositories of intracellular lipids used for energy production and membrane maintenance. During their formation in the membrane of the endoplasmic reticulum and through association with the plasma membrane, lipid droplets become armed with an array of proteins that are responsible for the organelle's structure, function, and signaling activities.

The compounds that concentrate in lipid droplets are not restricted to lipids. Proteomic characterization has revealed that proteins responsible for lipid transport, lipid metabolism, and droplet structure are physically associated with lipid droplets. Hydrophobic environmental chemicals, including PAHs, are another group of compounds that might concentrate within lipid droplets. Fluorescent PAHs, including B(a)P, as well as anthracene and fluoranthene, concentrate in fungal lipid droplets sequestering these noxious compounds (pollutant dissipation) and perhaps metabolize them to less toxic derivatives.

Here we report that BDS-associated fluorescence and a) a red fluorescent cholesterol analog or b) a transfected plasmid coding for a fluorescent lipid droplet surface protein co-localize within lipid droplets in human bronchoepithelial cells (BEAS-2B). BDS-exposed mouse adipocytes (3T3-L1) and alveolar macrophages (MH-S) develop equivalent fluorescence responses, further supporting the coalescence of combustion-derived PAHs within lipid droplets.

Microarray (BEAS-2B) and qRT-PCR (BEAS-2B, MH-S) results revealed a time-dependent up-regulation of Phase I biotransformation enzyme (CYP1A1, CYP1B1 and ALDH3A1) and Nrf2-mediated oxidative stress response (DNAJB6, FOS, HMOX1)genes in BDS-exposed cells.

Thus, these experiments demonstrate that combustion-derived PAHs adsorbed onto inhalable ambient particles are concentrated in lipid droplets of respiratory system cells. These PAHs concomitantly activate xenobiotic metabolism pathways that potentiate PAH toxicity and cellular responses to oxidative stress. These results recently have been published in detail (3).

(1) Environ Health Perspect 2001;109:965-971.

(2) Environ Health Perspect 2005;113:956-963.

(3) Am J Respir Cell Mol Biol 2008;38:532-540.