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.

Abstract. This is the second study in a three-part study designed to demonstrate dynamic entanglements among gaseous organic compounds (VOCs), 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, both in the dark and in sunlight. 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 living receptors. 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. Our exposure systems permit side-by-side, gas-only- and 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 for either gases or PM. In Part 1 (Ebersviller et al., 2012a), we demonstrated the existence of PM "effect modification" (NAS, 2004) for the case of a single gas-phase toxicant and an inherently non-toxic PM (mineral oil aerosol, MOA). That is, in the presence of the single gas-phase toxicant in the dark, the initially non-toxic PM became toxic to lung cells in the PM-only-biological exposure system. In this Part 2 study, we used sunlit-reactive systems to create a large variety of gas-phase toxicants from a complex mixture of oxides of nitrogen and 54 VOCs representative of those measured in US city air. In these mostly day-long experiments, we have designated the period in the dark just after injection (but before sunrise) as the "Fresh" condition and the period in the dark after sunset as the "Aged" condition. These two conditions were used to expose cells and to collect chemical characterization samples. We used the same inherently non-toxic PM from the Part 1 study as the target PM for "effect modification". Fortunately, in the absence of "seed particles", the complex highly-reactive VOC system used does not create any secondary aerosol in situ. All PM present in these tests were, therefore, introduced by injection of MOA to serve as PM-to-be-modified by the gaseous environment. PM addition was only done during dark periods, either before or after the daylight period. The purpose of this design is to test if a non-toxic PM becomes toxic in initially unreacted ("Fresh"), or in reacted ("Aged") complex VOC conditions. To have a complete design, we also tested the effects of clean air and the same VOC conditions, but without introducing any PM. Thus, there were six exposure treatment conditions that were evaluated with the side-by-side, gas-only- and PM-only-effects exposure systems; five separate chamber experiments were performed: two with clean air and three with the complex VOC/NOx mixture. For all of these experiments and exposures, chemical composition data and matching biological effects results for two end-points were compared. Chemical measurements demonstrate the temporal evolution of oxidized species, with a corresponding increase in toxicity observed from exposed cells. The largest increase in gas-phase toxicity was observed in the two "Aged" VOC exposures. The largest increase in particle-phase toxicity was observed in the "Aged" VOC exposure with the addition of PM after sunset. These results are a clear demonstration that the findings from Part 1 can be extended to the complex urban oxidized environment. This further demonstrates that the atmosphere itself cannot be ignored as a source of toxic species when establishing the risks associated with exposure to PM. Because gases and PM are transported and deposited differently within the atmosphere and lungs, these results have significant consequences. In the next (and final) part of the study, testing is further applied to systems with real diesel exhaust, including primary PM from a vehicle operated with different types of diesel fuel.

Air pollution consists of highly variable and complex mixtures recognized as major contributors to morbidity and mortality worldwide. The vast number of chemicals, coupled with limitations surrounding epidemiological and animal studies, has necessitated the development of new approach methods (NAMs) to evaluate air pollution toxicity. These alternative approaches include in vitro (cell-based) models, wherein toxicity of test atmospheres can be evaluated with increased efficiency compared to in vivo studies. In vitro exposure systems have recently been developed with the goal of evaluating air pollutant-induced toxicity; though the specific design parameters implemented in these NAMs-based studies remain in flux. This review aims to outline important design parameters to consider when using in vitro methods to evaluate air pollutant toxicity, with the goal of providing increased accuracy, reproducibility, and effectiveness when incorporating in vitro data into human health evaluations. This review is unique in that experimental considerations and lessons learned are provided, as gathered from first-hand experience developing and testing in vitro models coupled to exposure systems. Reviewed design aspects include cell models, cell exposure conditions, exposure chambers, and toxicity endpoints. Strategies are also discussed to incorporate in vitro findings into the context of in vivo toxicity and overall risk assessment.

RNAs are more susceptible to modifications than DNA, and chemical modifications in RNA have an effect on their structure and function. This study aimed to characterize chemical effects on total RNA in human A549 lung cells after exposure to elevated levels of major secondary air pollutants commonly found in urban locations, including ozone (O3), acrolein (ACR) and methacrolein (MACR). Enzyme-linked immunosorbent assays (ELISA) were used to measure levels of interleukin (IL)-8 in the growth media and 8-oxoguanine (8OG) levels in total cellular RNA, and lactate dehydrogenase (LDH) in the growth media was measured by a coupled enzymatic assay. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to measure levels of microRNA 10b (miR-10b). The study found that 1-h exposure to all tested pollutant mixtures consistently caused significant increases in the levels of 8OG in total RNA. In the case of 4 ppm O3 exposures, measured levels of IL-8, LDH and miR-10b each showed consistent trends between two independent trials, but varied among these three targets. After 1-h exposures to an ACR+MACR mixture, measured levels of IL-8, LDH and miR-10b showed variable results. For mixtures of O3+ACR+MACR, IL-8 measurements showed no change; miR-10b and LDH showed variable results. The results indicate that short-term high-concentration exposures to air pollution can cause RNA chemical modifications. Chemical modifications in RNAs could represent more consistent markers of cellular stress relative to other inflammation markers, such as IL-8 and LDH, and provide a new biomarker endpoint for mechanistic studies in toxicity of air pollution exposure.

VITROCELL systems permit cell exposures at the air-liquid interface (ALI); however, there are inconsistent methodologies in the literature for their operation. Some studies find that exposure to air (vehicle control) induced cytotoxicity relative to incubator controls; others do not mention if any cytotoxicity was encountered. We sought to test whether temperature and relative humidity (temp/RH) influence cytotoxicity with an unmodified (conditions A & B) and modified (condition C) VITROCELL 6 CF with temp/RH controls to permit conditioning of the sampled air-flow. We exposed BEAS-2B cells for 1 h to air and measured viability (WST-1 cell proliferation assay) and lactate dehydrogenase (LDH) release 6 h post-exposure. Relative to controls, cells exposed to air at (A) 22 °C and 18% RH had a 47.9% ± 3.2% ( < 0.0001) reduction in cell viability and 10.7% ± 2.0% ( < 0.0001) increase in LDH release (B) 22 °C and 55% RH had a 40.3% ± 5.8% ( < 0.0001) reduction in cell viability and 2.6% ± 2.0% ( = 0.2056) increase in LDH release, or (C) 37 °C and >75% RH showed no changes in cell viability and no increase in LDH release. Furthermore, cells exposed to air at 37 °C and >75% RH 24 h post-exposure showed no changes in viability or LDH release relative to incubator controls. Thus, reductions in cell viability were induced under conditions used typically in the literature (conditions A & B). However, our modifications (condition C) overcome this shortcoming by preventing cell desiccation; regulating temp/RH is essential for conducting adequate ALI exposures.

Abstract. This is the second study in a three-part study designed to demonstrate dynamic entanglements among gaseous organic compounds (VOCs), 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, both in the dark and in sunlight. To the traditional chemical and physical characterizations of gas and PM, we added new measurements of gas-only-and PM-onlybiological effects, using cultured human lung cells as model living receptors. 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. Our exposure systems permit side-by-side, gas-only-and 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 for either gases or PM.In Part 1 (Ebersviller et al., 2012a), we demonstrated the existence of PM "effect modification" (NAS, 2004) for the case of a single gas-phase toxicant and an inherently nontoxic PM (mineral oil aerosol, MOA). That is, in the presence of the single gas-phase toxicant in the dark, the initially non-toxic PM became toxic to lung cells in the PM-onlybiological exposure system. In this Part 2 study, we used sunlit-reactive systems to create a large variety of gas-phase toxicants from a complex mixture of oxides of nitrogen and 54 VOCs representative of those measured in US city air. In these mostly day-long experiments, we have designated the period in the dark just after injection (but before sunrise) as the "Fresh" condition and the period in the dark after sunset as the "Aged" condition. These two conditions were used to expose cells and to collect chemical characterization samples. We used the same inherently non-toxic PM from the Part 1 study as the target PM for "effect modification". Fortunately, in the absence of "seed particles", the complex highly-reactive VOC system used does not create any secondary aerosol in situ. All PM present in these tests were, therefore, introduced by injection of MOA to serve as PM-to-be-modified by the gaseous environment. PM addition was only done during dark periods, either before or after the daylight period. The purpose of this design is to test if a non-toxic PM becomes toxic in initially unreacted ("Fresh"), or in reacted ("Aged") complex VOC conditions. To have a complete design, we also tested the effects of clean air and the same VOC conditions, but without introducing any PM. Thus, there were six exposure treatment conditions that were evaluated with the side-by-side, gas-only-and PMonly-effects exposure systems; five separate chamber experiments were performed: two with clean air and three with the complex VOC/NO x mixture.For all of these experiments and exposures, chemical composition data and matching biological effects results for two end-points were compared. Chemic...