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Previous studies have indicated the roles of both organic compounds and metals in driving the cellular generation of reactive oxygen species (ROS); however, their contribution has not been adequately quantified using mechanistic approaches. We developed a novel fractionation scheme for the stepwise removal of various classes of organic compounds and metals using a combination of solid phase extraction columns. We applied this fractionation scheme to 10 PM 2.5 samples collected from the midwestern United States. Because both watersoluble organic carbon (WSOC) and Fe have shown good correlations with cellular ROS, we separated them into different fractions and measured their ability to generate ROS in rat alveolar macrophages. Most of the PM 2.5 cellular ROS was attributed to the metallic fraction. To further explore the reason for the correlation of WSOC with ROS, we investigated the water solubility of Fe by measuring the total Fe in PM 2.5 samples. The water-soluble fraction of Fe was tightly correlated with WSOC (r ≥ 0.69), indicating WSOC may have an additional role in cellular oxidative potential, probably through complexation of Fe, in enhancing its water solubility and macrophage ROS activity. This work reveals the role of both Fe and organic compounds through different mechanisms in contributing to PM 2.5 -driven cellular ROS.

We investigated the influence of biomass burning (BURN), Diwali fireworks, and fog events on the ambient fine particulate matter (PM 2.5 ) oxidative potential (OP) during the postmonsoon (PMON) and winter season in Delhi, India. The real-time hourly averaged OP (based on a dithiothreitol assay) and PM 2.5 chemical composition were measured intermittently from October 2019 to January 2020. The peak extrinsic OP (OP v : normalized by the volume of air) was observed during the winter fog (WFOG) (5.23 ± 4.6 nmol•min −1 •m −3 ), whereas the intrinsic OP (OP m ; normalized by the PM 2.5 mass) was the highest during the Diwali fireworkinfluenced period (29.4 ± 18.48 pmol•min −1 •μg −1 ). Source apportionment analysis using positive matrix factorization revealed that traffic + resuspended dust-related emissions (39%) and secondary sulfate + oxidized organic aerosols (38%) were driving the OP v during the PMON period, whereas BURN aerosols dominated (37%) the OP v during the WFOG period. Firework-related emissions became a significant contributor (∼32%) to the OP v during the Diwali period (4 day period from October 26 to 29), and its contribution peaked (72%) on the night of Diwali. Discerning the influence of seasonal and episodic sources on health-relevant properties of PM 2.5 , such as OP, could help better understand the causal relationships between PM 2.5 and health effects in India.

Abstract. Several automated instruments exist to measure the acellular oxidative potential (OP) of ambient particulate matter (PM). However, cellular OP of the ambient PM is still measured manually, which severely limits the comparison between two types of assays. Cellular assays could provide a more comprehensive assessment of the PM-induced oxidative stress, as they incorporate more biological processes involved in the PM-catalyzed reactive oxygen species (ROS) generation. Considering this need, we developed a semi-automated instrument, the first of its kind, for measuring the cellular OP based on a macrophage ROS assay using rat alveolar macrophages. The instrument named SCOPE – semi-automated instrument for cellular oxidative potential evaluation – uses dichlorofluorescein diacetate (DCFH-DA) as a probe to detect the OP of PM samples extracted in water. SCOPE is capable of analyzing a batch of six samples (including one negative and one positive control) in 5 h and is equipped to operate continuously for 24 h with minimal manual intervention after every batch of analysis, i.e., after every 5 h. SCOPE has a high analytical precision as assessed from both positive controls and ambient PM samples (coefficient of variation (CoV)<17 %). The results obtained from the instrument were in good agreement with manual measurements using tert-butyl hydroperoxide (t-BOOH) as the positive control (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 SCOPE 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, polycyclic aromatic hydrocarbons (PAHs) and inorganic salts, commonly known to be present in ambient PM. This dataset is potentially useful in future studies to apportion the contribution of key chemical species in the overall cellular OP of ambient PM.

Abstract. Several automated instruments exist to measure the acellular oxidative potential (OP) of ambient particulate matter (PM). However, cellular OP of the ambient PM is still measured manually, which severely limits the comparison between two types of assays. Cellular assays could provide a more comprehensive assessment of the PM-induced oxidative stress, as they incorporate more biological processes involved in the PM-catalyzed reactive oxygen species (ROS) generation. Considering this need, we developed a first of its kind semi-automated instrument for measuring the cellular OP based on a macrophage ROS assay using rat alveolar macrophages. The instrument named SCOPE – Semi-automated instrument for Cellular Oxidative Potential Evaluation, uses dichlorofluorescein diacetate (DCFH-DA) as a probe to detect the OP of PM samples extracted in water. SCOPE is capable of analyzing a batch of six samples (including one negative and one positive control) in five hours and is equipped to operate continuously for 24-hours with minimal manual intervention after every batch of analysis, i.e., after every five hours. SCOPE has a high analytical precision as assessed from both positive controls and ambient PM samples (CoV < 17 %). The results obtained from the instrument were in good agreement with manual measurements using tert-Butyl hydroperoxide (t-BOOH) as the positive control (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 SCOPE 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. This dataset is potentially useful in future studies to apportion the contribution of key chemical species in the overall cellular OP of ambient PM.

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