Assessing cellular responses to ambient PM2.5 in the Midwest USA using a Semi-automated Instrument for Cellular Oxidative Potential Evaluation (SCOPE) and flow cytometric analysis

In recent years, an increasing number of studies have focused on the Oxidative Potential (OP) measurements as a proxy for ambient particulate matter (PM) toxicity. Many of these measurements are conducted in chemical assays which are sensitive to only certain chemical components of PM and include only a single pathway of ROS generation. Hence, there is a need to establish the biological relevance of the chemical OP assays by comparing their responses with the cellular responses. In this regard, we have adopted a two-pronged approach. Given the labor and time-consuming protocols of the cellular processes, first we developed a first of its kind Semi-automated Instrument for Cellular Oxidative Potential Evaluation (SCOPE) of water-soluble extracts of ambient PM. The instrument is equipped to operate continuously for 24-hours with minimal manual intervention and has a high analytical precision (CoV <20% for positive controls and ambient PM sample). The results obtained from the instrument were in good agreement with manual measurements using t-BOOH (slope = 0.83 for automated vs. manual, R2 = 0.99) and ambient samples (slope = 0.83, R2 = 0.71). We further demonstrated the ability of this instrument to analyze a large number of both ambient and laboratory samples, and developed a dataset on the intrinsic cellular OP of several compounds, such as metals, quinones, PAHs and inorganic salts, commonly known to be present in ambient PM. Second, we measured various cellular responses (cytotoxicity, genotoxicity and oxidative stress using SCOPE) of a large number of ambient PM samples (N >200) collected in five locations (Chicago, Champaign, Bondville, St. Louis, Indianapolis) in the Midwest region of USA using NR8383 rat alveolar macrophage cells. We used a combination of Propidium Iodide/AnnexinV FITC assays for apoptosis and necrosis detection, and H2AX assay for measuring DNA double strand damage using a flow cytometer. Preliminary results show that there is a good correlation (R2~0.5) between oxidative stress (measured after 24 h incubation) and apoptosis and DNA damage and a poor correlation between oxidative stress and necrosis. We plan to conduct a comprehensive comparison among all the endpoints to systematically understand the role of oxidative stress in the PM2.5 toxicological pathways.