Background: Air pollution contributes significantly to global increases in mortality, particularly within urban environments. Limited knowledge exists on the mechanisms underlying health effects resulting from exposure to pollutant mixtures similar to those occurring in ambient air. In order to clarify the mechanisms underlying exposure effects, toxicogenomic analyses are used to evaluate genomewide transcript responses and map these responses to molecular networks.Objectives: We compared responses induced by exposure to primary pollutants and photochemically altered (PCA) pollutant mixtures representing urban atmospheres to test our hypothesis that exposures to PCA pollutants would show increased modulation of inflammation-associated genes and pathways relative to primary air pollutants.Methods: We used an outdoor environmental irradiation chamber to expose human lung epithelial cells to mixtures representing either primary or PCA pollutants for 4 hr. Transcriptional changes were assessed using microarrays and confirmed using quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR) on a subset of genes.Results: We found a large difference in the cellular responses to the two pollutant exposures: Primary air pollutants altered the expression levels of 19 genes, whereas PCA pollutants altered 709 genes. Functional and molecular analyses of the altered genes revealed novel pathways, such as hepatocyte nuclear factor 4α, potentially regulating the pollutant responses. Chemical component analysis characterized and confirmed the photochemical transformation of primary air pollutants into PCA air pollutants.Conclusions: Our study shows that the photochemical transformation of primary air pollutants produces altered mixtures that cause significantly greater biological effects than the primary pollutants themselves. These findings suggest that studying individual air pollutants or primary pollutant mixtures may greatly underestimate the adverse health effects caused by air pollution.

One of the most widely used in vitro particulate matter (PM) exposures methods is the collection of PM on filters, followed by resuspension in a liquid medium, with subsequent addition onto a cell culture. To avoid disruption of equilibria between gases and PM, we have developed a direct in vitro sampling and exposure method (DSEM) capable of PM-only exposures. We hypothesize that the separation of phases and post-treatment of filter-collected PM significantly modifies the toxicity of the PM compared to direct deposition, resulting in a distorted view of the potential PM health effects. Controlled test environments were created in a chamber that combined diesel exhaust with an urban-like mixture. The complex mixture was analyzed using both the DSEM and concurrently-collected filter samples. The DSEM showed that PM from test atmospheres produced significant inflammatory response, while the resuspension exposures at the same exposure concentration did not. Increasing the concentration of resuspended PM sixteen times was required to yield measurable IL-8 expression. Chemical analysis of the resuspended PM indicated a total absence of carbonyl compounds compared to the test atmosphere during the direct-exposures. Therefore, collection and resuspension of PM into liquid modifies its toxicity and likely leads to underestimating toxicity.

This is the first of a three-part study designed to demonstrate dynamic entanglements among gaseous organic compounds (VOC), particulate matter (PM), and their subsequent potential biological effects. We study these entanglements in increasingly complex VOC and PM mixtures in urban-like conditions in a large outdoor chamber. To the traditional chemical and physical characterizations of gas and PM, we added new measurements of gas-only- and PM-only-biological effects, using cultured human lung cells as model indicators. These biological effects are assessed here as increases in cellular damage or expressed irritation (i.e., cellular toxic effects) from cells exposed to chamber air relative to cells exposed to clean air. The exposure systems permit gas-only- or PM-only-exposures from the same air stream containing both gases and PM in equilibria, i.e., there are no extractive operations prior to cell exposure. Our simple experiments in this part of the study were designed to eliminate many competing atmospheric processes to reduce ambiguity in our results. Simple volatile and semi-volatile organic gases that have inherent cellular toxic properties were tested individually for biological effect in the dark (at constant humidity). Airborne mixtures were then created with each compound and PM that has no inherent cellular toxic properties for another cellular exposure. Acrolein and p-tolualdehyde were used as model VOCs and mineral oil aerosol (MOA) was selected as a surrogate for organic-containing PM. MOA is appropriately complex in composition to represent ambient PM, and it exhibits no inherent cellular toxic effects and thus did not contribute any biological detrimental effects on its own. Chemical measurements, combined with the responses of our biological exposures, clearly demonstrate that gas-phase pollutants can modify the composition of PM (and its resulting detrimental effects on lung cells) – even if the gas-phase pollutants are not considered likely to partition to the condensed phase: the VOC-modified-PM showed significantly more damage and inflammation to lung cells than did the original PM. Because gases and PM are transported and deposited differently within the atmosphere and the lungs, these results have significant consequences. For example, current US policies for research and regulation of PM do not recognize this “effect modification” phenomena (NAS, 2004). These results present an unambiguous demonstration that – even in these simple mixtures – physical and thermal interactions alone can cause a modification of the distribution of species among the phases of airborne pollution mixtures and can result in a non-toxic phase becoming toxic due to atmospheric thermal processes only. Subsequent work extends the simple results reported here to systems with photochemical transformations of complex urban mixtures and to systems with diesel exhaust produced by different fuels.

Abstract. This is the first of a three-part study designed to demonstrate dynamic entanglements among gaseous organic compounds (VOC), particulate matter (PM), and their subsequent potential biological effects. We study these entanglements in increasingly complex VOC and PM mixtures in urban-like conditions in a large outdoor chamber. To the traditional chemical and physical characterizations of gas and PM, we added new measurements of biological effects, using cultured human lung cells as model indicators. These biological effects are assessed here as increases in cellular damage or expressed irritation (i.e., cellular toxic effects) from cells exposed to chamber air relative to cells exposed to clean air. The exposure systems permit virtually gas-only- or PM-only-exposures from the same air stream containing both gases and PM in equilibria, i.e., there are no extractive operations prior to cell exposure. Our simple experiments in this part of the study were designed to eliminate many competing atmospheric processes to reduce ambiguity in our results. Simple volatile and semi-volatile organic gases that have inherent cellular toxic properties were tested individually for biological effect in the dark (at constant humidity). Airborne mixtures were then created with each compound to which we added PM that has no inherent cellular toxic properties for another cellular exposure. Acrolein and p-tolualdehyde were used as model VOCs and mineral oil aerosol (MOA) was selected as a surrogate for organic-containing PM. MOA is appropriately complex in composition to represent ambient PM, and exhibits no inherent cellular toxic effects and thus did not contribute any biological detrimental effects on its own. Chemical measurements, combined with the responses of our biological exposures, clearly demonstrate that gas-phase pollutants can modify the composition of PM (and its resulting detrimental effects on lung cells). We observed that, even if the gas-phase pollutants are not considered likely to partition to the condensed phase, the VOC-modified-PM showed significantly more damage and inflammation to lung cells than did the original PM. Because gases and PM are transported and deposited differently within the atmosphere and the lungs, these results have significant consequences for a wide range of people. For example, current US policies for research and regulation of PM do not recognize this "effect modification" phenomena (NAS, 2004). These results present an unambiguous demonstration that – even in these simple mixtures – physical and thermal interactions alone can cause a modification of the distribution of species among the phases of airborne pollution mixtures that can result in a non-toxic phase becoming toxic due to atmospheric thermal processes only. Subsequent work (described in companion papers) extends the simple results reported here to systems with photochemical transformations of complex urban mixtures and to systems with diesel exhaust produced by different fuels.

Context EpiAirway™ 3-D constructs are human-derived cell cultures of differentiated airway epithelial cells that may represent a more biologically relevant model of the human lung. However, limited information is available of its utility for exposures to air pollutants at the air-liquid interface (ALI). Objective To assess the biological responses of EpiAirway™ cells in comparison to the responses of A549 human alveolar epithelial cells after exposure to air pollutants at ALI. Methods Cells were exposed to filtered air, 400ppb of ozone (O3) or a photochemically-aged Synthetic Urban Mixture (SynUrb54) consisting of hydrocarbons, nitrogen oxides, O3, and other secondary oxidation products for 4 h. Basolateral supernatants and apical washes were collected at 9 and 24 h post-exposure. We assessed cytotoxicity by measuring lactate dehydrogenase (LDH) release into the culture medium and apical surface. Interleukin 6 (IL-6) and interleukin 8 (IL-8) proteins were measured in the culture medium and in the apical washes to determine the inflammatory response after exposure. Results Both O3 and SynUrb54 significantly increased basolateral levels of LDH and IL-8 in A549 cells. No significant changes in LDH and IL-8 levels were observed in the EpiAirway™ cells, however, IL-6 in the apical surface was significantly elevated at 24 h after O3 exposure. Conclusion LDH and IL-8 are robust endpoints for assessing toxicity in A549 cells. The EpiAirway™ cells show minimal adverse effects after exposure suggesting that they are more toxicologically resistant compared to A549 cells. Higher concentrations or longer exposure times are needed to induce effects on EpiAirway™ cells.