Understanding the Early Biological Effects of Isoprene-Derived Particulate Matter Enhanced by Anthropogenic Pollutants.

INTRODUCTION: Airborne fine particulate matter (PM2.5; particulate matter ≤ 2.5 µm in aerodynamic diameter) plays a key role in air quality, climate, and public health. Globally, the largest mass fraction of PM2.5 is organic, dominated by secondary organic aerosol (SOA) formed from atmospheric oxidation of volatile organic compounds (VOCs). Isoprene from vegetation is the most abundant nonmethane VOC emitted into Earth's atmosphere. Isoprene has been recently recognized as one of the major sources of global SOA production that is enhanced by the presence of anthropogenic pollutants, such as acidic sulfate derived from sulfur dioxide (SO2), through multiphase chemistry of its oxidation products. Considering the abundance of isoprene-derived SOA in the atmosphere, understanding mechanisms of adverse health effects through inhalation exposure is critical to mitigating its potential impact on public health. Although previous studies have examined the toxicological effects of certain isoprene-derived gas-phase oxidation products, to date, no systematic studies have examined the potential toxicological effects of isoprene-derived SOA, its constituents, or its SOA precursors on human lung cells. SPECIFIC AIMS: The overall objective of this study was to investigate the early biological effects of isoprene-derived SOA and its subtypes on BEAS-2B cells (a human bronchial epithelial cell line), with a particular focus on the alteration of oxidative stress- and inflammation-related genes. To achieve this objective, there were two specific aims.1. Examine toxicity and early biological effects of SOA derived from the photochemical oxidation of isoprene, considering both urban and downwind-urban types of chemistry.2. Examine toxicity and early biological effects of SOA derived directly from downstream oxidation products of isoprene (i.e., epoxides and hydroperoxides). METHODS: Isoprene-derived SOA was first generated by photooxidation of isoprene under natural sunlight in the presence of nitric oxide (NO) and acidified sulfate aerosols. Experiments were conducted in a 120-m3 outdoor Teflon-film chamber located on the roof of the Gillings School of Global Public Health, University of North Carolina at Chapel Hill (UNC-Chapel Hill). BEAS-2B cells were exposed to chamber- generated isoprene-derived SOA using the Electrostatic Aerosol in Vitro Exposure System (EAVES). This approach allowed us to generate atmospherically relevant compositions of isoprene-derived SOA and to examine its toxicity through in vitro exposures at an air-liquid interface, providing a more biologically relevant exposure model. Isoprene-derived SOA samples were also collected, concurrently with EAVES sampling, onto Teflon membrane filters for in vitro resuspension exposures and for analysis of aerosol chemical composition by gas chromatography/electron ionization-quadrupole mass spectrometry (GC/EI-MS) with prior trimethylsilylation and ultra-performance liquid-chromatography coupled to high-resolution quadrupole time-of-flight mass spectrometry equipped with electrospray ionization (UPLC/ESI-HR-QTOFMS). Isoprene-derived SOA samples were also analyzed by the dithiothreitol (DTT) assay in order to characterize their reactive oxygen species (ROS)-generation potential.Organic synthesis of known isoprene-derived SOA precursors, which included isoprene epoxydiols (IEPOX), methacrylic acid epoxide (MAE), and isoprene-derived hydroxyhydroperoxides (ISOPOOH), was conducted in order to isolate major isoprene-derived SOA formation pathways from each other and to determine which of these pathways (or SOA types) is potentially more toxic. Since IEPOX and MAE produce SOA through multiphase chemistry onto acidic sulfate aerosol, dark reactive uptake experiments of IEPOX and MAE in the presence of acidic sulfate aerosol were performed in a 10-m3 flexible Teflon indoor chamber at UNC-Chapel Hill. Since the generation of SOA from ISOPOOH (through a non-IEPOX route) requires a hydroxyl radical (•OH)-initiated oxidation, ozonolysis of tetramethylethylene (TME) was used to form the needed •OH radicals in the indoor chamber. The resultant low-volatility multifunctional hydroperoxides condensed onto nonacidified sulfate aerosol, yielding the ISOPOOH-derived SOA needed for exposures. Similar to the outdoor chamber SOAs, IEPOX, MAE- and ISOPOOH-derived SOAs were collected onto Teflon membrane filters and were subsequently chemically characterized by GC/EI-MS and UPLC/ESI-HR-QTOFMS as well as for ROS-generation potential using the DTT assay. These filters were also used for resuspension in vitro exposures.By conducting gene expression profiling, we provided mechanistic insights into the potential health effects of isoprene-derived SOA. First, gene expression profiling of 84 oxidative stress- and 249 inflammation-associated human genes was performed for cells exposed to isoprene-derived SOA generated in our outdoor chamber experiments in EAVES or by resuspension. Two pathway-focused panels were utilized for this purpose: (1) nCounter GX Human Inflammation Kit comprised of 249 human genes (NanoString), and (2) Human Oxidative Stress Plus RT2 Profiler PCR Array (Qiagen) comprised of 84 oxidative stress-associated genes. We compared the gene expression levels in cells exposed to SOA generated in an outdoor chamber from photochemical oxidation of isoprene in the presence of NO and acidified sulfate seed aerosol to cells exposed to a dark control mixture of isoprene, NO, and acidified sulfate seed aerosol to isolate the effects of the isoprene-derived SOA on the cells using the EAVES and resuspension exposure methods. Pathway-based analysis was performed for significantly altered genes using the ConsensusPathDB database, which is a database system for the integration of human gene functional interactions to provide biological pathway information for a gene set of interest. Pathway annotation was performed to provide biological pathway information for each gene set. The gene-gene interaction networks were constructed and visualized using the GeneMANIA Cytoscape app (version 3.4.1) to predict the putative function of altered genes. Lastly, isoprene-derived SOA collected onto filters was used in resuspension exposures to measure select inflammatory biomarkers, including interleukin 8 (IL-8) and prostaglandin-endoperoxide synthase 2 (PTGS2) genes, in BEAS-2B cells to ensure that effects observed from EAVES exposures were attributable to particle-phase organic products. Since EAVES and resuspension exposures compared well, gene expression profiling for IEPOX-, MAE- and ISOPOOH-derived SOA were conducted using only resuspension exposures. RESULTS AND CONCLUSIONS: Chemical characterization coupled with biological analyses show that atmospherically relevant compositions of isoprene-derived SOA alter the levels of 41 oxidative stress-related genes. Of the different composition types of isoprene-derived SOA, MAE- and ISOPOOH-derived SOA altered the greatest number of genes, suggesting that carbonyl and hydroperoxide functional groups are oxidative stress promoters. Taken together, the different composition types accounted for 34 of the genes altered by the total isoprene-derived SOA mixture, while 7 remained unique to the total mixture exposures, indicating that there is either a synergistic effect of the different isoprene-derived SOA components or an unaccounted component in the mixture.The high-oxides of nitrogen (NOx) regime, which yielded MAE- and methacrolein (MACR)-derived SOA, had a higher ROS-generation potential (as measured by the DTT assay) than the low- NOx regime, which included IEPOX- and isoprene-derived SOA. However, ISOPOOH-derived SOA, which also formed in the low- NOx regime, had the highest ROS-generation potential, similar to 1,4-naphthoquinone (1,4-NQ). This suggests that aerosol-phase organic peroxides contribute significantly to particulate matter (PM) oxidative potential. MAE- and MACR- derived SOA showed equal or greater ROS-generation potential than was reported in prior UNC-Chapel Hill studies on diesel exhaust PM, highlighting the importance of a comprehensive investigation of the toxicity of isoprene-derived SOA. Notably, ISOPOOH-derived SOA was one order of magnitude higher in ROS-generation potential than diesel exhaust particles previously examined at UNC-Chapel Hill. As an acellular assay, the DTT assay may not be predictive of oxidative stress; therefore, we also focused on the gene expression results from the cellular exposures.We have demonstrated that the nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and the redox-sensitive activation protein-1 (AP-1) transcription factor networks have been significantly altered upon exposure to isoprene-derived SOA. The identification of Nrf2 pathway in cells exposed to isoprene-derived SOA is in accordance with our findings using the DTT assay, which measures the thiol reactivity of PM samples as a surrogate for their ROS-generation potential. Specifically, our results point to the cysteine-thiol modifications within cells that lead to activation of Nrf2-related gene expression.However, based on our gene expression results showing no clear relationship between DTT activity and the number of altered oxidative stress-related genes, the DTT activity of isoprene-derived SOA may not be directly indicative of toxicity relative to other SOA types. While activation of Nrf2-associated genes has been identified with responses to oxidative stress and linked to traffic related air pollution exposure in both toxicological and epidemiological studies, their implicit involvement in this study suggests that activation of Nrf2-related gene expression may occur with exposures to all sorts of PM types.By controlling the exposure time, method, and dose we demonstrated that among the SOA derived from previously identified individual precursors of isoprene-derived SOA, ISOPOOH-derived SOA alters more oxidative stress related genes than does IEPOX-derived SOA, but fewer than MAE-derived SOA. This suggests that the composition of MAE-derived SOA may be the greatest contributor to alterations of oxidative stress-related gene expression observed due to isoprene-derived SOA exposure. Further study on induced levels of protein expression and specific toxicological endpoints is necessary to determine if the observed gene expression changes lead to adverse health effects. In addition, such studies have implications for pollution-control strategies because NOx and SO2 are controllable pollutants that can alter the composition of SOA, and in turn alter its effects on gene expression. The mass fraction of different components of atmospheric isoprene derived SOA should be considered, but altering the fraction of high- NOx isoprene-derived SOA (e.g., MAE derived SOA) may yield greater changes in gene expression than altering the fraction of low- NOx isoprene derived SOA types (ISOPOOH- or IEPOX-derived SOA). Finally, this study confirms that total isoprene-derived SOA alters the expression of a greater number of genes than does SOA derived from the tested precursors. This warrants further work to determine the underlying explanation for this observation, which may be uncharacterized components of isoprene-derived SOA or the potential for synergism between the studied components.

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