(167d) Soot Nanoparticles Promote Biotransformation, Oxidative Stress, and Inflammation in Murine Lungs

Urban ambient pollution particles are composed of a complex array of organic and inorganic components, often adsorbed to a carbonaceous core. Fine particles (PM2.5; aerodynamic diameter 0.1-2.5 µm) contribute to ambient pollution and to cardiopulmonary morbidity. PM2.5 exposure correlates with increased cardiopulmonary and lung cancer mortality, as well as increased risk of respiratory and cardiovascular disease. Exposure to the coarse (PM10; aerodynamic diameter 2.5-10.0 µm) particulate component of air pollution generates an increase in the circulating inflammatory cytokines, IL-1â, IL-6, IL-8. Following comparable exposure in vitro, alveolar macrophages and bronchial epithelial cells produced these same cytokines, suggesting that cytokines produced by respiratory cells can act locally and systemically. To date, ultrafine particles (PM 0.1; aerodynamic diameter < 0.1 ìm) and nanoparticles (aerodynamic diameter < 0.150 ìm) have received little attention from regulatory agencies, but in numerous experimental settings have been found to elicit a range of toxicological effects (often more severe than those found with comparable exposures to fine particles), including inflammatory cell infiltration and impaired macrophage phagocytosis.

The surface characteristics and crystalline structure of ambient particles can be major determinants of pulmonary inflammation and injury. Individually or in concert, members of the complex chemical array adsorbed to particles also can promote disease processes. The aqueous, inorganic, transition metal-enriched fraction of residual oil fly ash (ROFA), containing transition metals, induces edema, hemorrhage, and a profound inflammatory infiltrate in the lung. Diesel exhaust particles (DEPs) containing a variety of oxygen radical-generating quinones, polyaromatic hydrocarbons (PAHs), and metal species produce pulmonary inflammatory cell infiltration and inflammatory cytokine production by bronchial epithelial cells and alveolar macrophages. Most of the effects have been attributed to the PAH and quinone components of DEPs. A body of literature, both in vitro and in vivo, has accumulated characterizing the effects of the parent particles and their isolated constituents in humans, as well as in animals.

In urban areas, combustion of gasoline, diesel fuels, and industrial organics (simple aliphatics and/or fossil fuels) contributes significantly to the ambient PM 2.5 fraction and to the ultrafine particulate fraction. Incomplete combustion of low molecular weight hydrocarbons, as in the case of industrial flaring of fugitive volatiles, is also a real source of complex environmental particulate contamination. Furthermore, petrochemical-derived nanoparticles resulting, by accident or sabotage, from refinery or pipeline explosions and fires represent a real hazard both in the United States and abroad.

In the burning of hydrocarbons, radicals formed early in combustion interact forming PAHs, including carcinogens, from less complex structures. PAHs will aggregate into nanoparticles, which can extend into branched-chain structures (soots). We have previously characterized the product of incomplete combustion of 1,3-butadiene (BD), a high-volume aliphatic hydrocarbon by-product of petroleum refining that is used in the manufacture of synthetic rubber and other elastomers. This product, butadiene soot (BDS), is both a model mixture and a real-life example of PAH-laden, combustion-derived nanoparticles with potential for environmental contamination and for acute and/or chronic health effects.

BDS is an organic-rich mixture of 30-50 nm carbonaceous particles to which hundreds of PAH species, including benzo(a)pyrene [B(a)P] and other carcinogens, are adsorbed. In contrast to ROFA and DEPs, BDS is relatively oxygen- and metals-poor. Both, human bronchoepithelial cells and mouse alveolar macrophages display a distinct punctate blue, PAH-associated cytoplasmic fluorescence following exposure to BDS in vitro. The fluorescence localizes to cytoplasmic lipid droplets even as Phase I biotransformation enzymes are induced. Fluorescence does not develop if PAHs are extracted from the particles prior to cell exposure. Fluorescent cells contain the same spectrum of PAHs present in the parent BDS.

We hypothesized based on our in vitro studies (3) that inhalation of PAH-containing BDS will result in activation of aryl hydrocarbon receptor (AhR)-associated genes, as is the case with other PAH-rich mixtures (DEPs, cigarette smoke), will cause oxidative stress and will result in inflammatory changes, including inflammatory infiltrate and up-regulation of inflammatory cytokines, as is the case with DEP.

Female Balb/c mice exposed to BDS (5 mg/m3; 4 hr/day; 4 days) were sacrificed immediately or one day following final exposure; bronchoalveolar lavage fluid (BALF) was collected from the lungs; total RNA was extracted from one lung and histopathology performed on the other. Histopathology and BALF analysis revealed particle-laden macrophages in airways of BDS treated mice, accompanied by neutrophilia and epithelial damage. Microarray and qRT-PCR analyses revealed up-regulation of a) aryl hydrocarbon receptor (AhR)-responsive genes: AhR repressor (Ahrr) and cytochrome P450 IA1 and IB1(Cyp1a1, Cyp1b1); b) oxidative stress response genes: heme oxygenase 1 (Hmox1), nuclear factor erythroid-derived 2 like 2 (Nfe2l2), NADPH dehydrogenase quinone 1 (Nqo1), and glutathione peroxidase 2 (Gpx2); and c) pro-inflammatory genes: interleukin-6 (IL-6), C-X-C motif ligand 2 (Cxcl2; analog to human IL-8) and ligand 3 (Cxcl3), and granulocyte chemotactic protein (Cxcl6).

In conclusion, inhalation of PAH-rich, petrochemical combustion-derived nanoparticles causes airway inflammation and induces expression of AhR-associated and oxidative stress response genes, as seen in vitro, plus pro-inflammatory genes.