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. 2026 Sep 30;11(40):61434–61441. doi: 10.1021/acsomega.6c06562

Superoxide Release from Macrophages upon Exposure to Secondary Organic Aerosols, Biomass, and Plastic Burning Particulate Matter

Kasey C Edwards †, Ting Fang ‡,§,*, Rizana Salim †,∥, Caitlyn Cruz †, Sukriti Kapur †, Sachin S Gunthe ∥, Sergey A Nizkorodov †, Manabu Shiraiwa †,*
PMCID: PMC13670832  PMID: 42889634

Abstract

Oxidative stress induced by reactive oxygen species (ROS) is a key process for adverse health effects upon respiratory deposition of particulate matter (PM). Alveolar macrophages can release superoxide upon phagocytosis of PM through a process called the respiratory burst. The release of cellular ROS should depend on both PM composition and dose, but the dose–response relationship for various types of PM remains poorly quantified. Here we measure cellular superoxide production by macrophage cells exposed to secondary organic aerosols (SOA) derived from oxidation of limonene, toluene, and naphthalene, as well as PM generated from biomass burning and flaming combustion of five different types of plastics. We quantify cellular superoxide production over the span of 4 h after an initial exposure of macrophages to PM suspensions by applying a chemiluminescence assay combined with electron paramagnetic resonance. We then define the activation threshold as the dose at which cells exhibit a clear increase in superoxide production. Limonene SOA is found to induce the largest amount of superoxide release (∼32 μM) but has the largest activation threshold of 300 μg mL–1. Naphthalene and toluene SOA show smaller enhancement of superoxide production (∼2.5–7 μM) but have lower activation thresholds from 0.02 to 0.1 μg mL–1. Biomass burning PM does not show a clear activation threshold. Plastic burning PM shows a varied response depending on the type of plastic, with PM derived from poly­(ethylene terephthalate) (PET) having the highest superoxide production (∼12 μM). The activation thresholds for polystyrene (PS), low-density polyethylene (LDPE), and high-density polyethylene (HDPE) burning PM are very low (∼0.01 μg mL–1), while those for poly­(vinyl chloride) (PVC) and PET burning PM are substantially higher (50–100 μg mL–1). Our findings highlight the importance of employing dose–response relationships and superoxide activation thresholds, rather than single-dose comparisons, to evaluate and compare the oxidative stress potential of different PMs more accurately.

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1. Introduction

Inhalation of particulate matter (PM) is the cause of several millions of premature deaths per year, and prolonged exposure to PM can lead to respiratory and cardiovascular diseases. , PM is emitted directly into the atmosphere or generated in situ through the oxidation of volatile organic compounds (VOCs), forming secondary organic aerosols (SOA). One of the plausible mechanisms for PM to induce adverse health effects is oxidative stress mediated by reactive oxygen species (ROS). ROS include hydrogen peroxide (H2O2), superoxide (•O2 –), hydroxyl radical (•OH), and oxygen-centered organic radicals. , PM contains redox-active compounds, such as transition metals and quinones, which can undergo redox reactions to form ROS. SOA contain substantial amounts of peroxides that can generate ROS in the aqueous phase. , ROS are also generated by cells and activate cell signaling pathways that can lead to many different types of immune responses. While ROS are important on a cellular level for physiological processes, excess ROS can overwhelm the natural antioxidant response, resulting in oxidative stress by damaging biological molecules such as lipids, proteins, and DNA. ,

Alveolar macrophages are the first cellular responders to inhaled PM. , As part of the immune response, macrophages can engulf PM through phagocytosis. Simultaneously, phagocytes will uptake large amounts of oxygen to generate large quantities of ROS (H2O2 and •O2 –) in a process known as a respiratory burst. ,, The respiratory burst is driven by the enzyme NADPH oxidase, which transports electrons from NADPH to O2, producing •O2 –. A study by Fang et al. (2022) showed that, through NADPH oxidase activation, •O2 – is released from macrophages exposed to isoprene SOA and 9,10-phenanthrenequinone. Studies have shown that fine PM can also activate ROS generation, which can lead to autophagy dysfunction. Ambient PM has been shown to induce proinflammatory cytokine responses, which are secreted by macrophages. , The macrophage response has been shown to be dependent on the size and composition of PM. , Macrophages overloaded with PM can result in an impairment of their phagocytic activity. , Previous studies have found positive associations between cellular ROS levels and cytotoxicity and other biological effects from SOA exposure. ,

Quantification of cellular ROS release from exposure to various types of SOA and primary PM is still very limited. Here we investigate the effect of various types of PM on the release of •O2 – by macrophages. RAW 264.7 macrophage cells are exposed to SOA and PM generated by biomass burning and plastic combustion. We generate SOA from limonene as a representative biogenic VOC and from toluene and naphthalene as representative anthropogenic VOCs. Biomass burning PM is of growing health concern because of the increase of wildfires due to global environmental change. Open burning of plastic wastes is especially common in developing countries, and the health effects of PM are still largely unknown. We quantify total •O2 – generation by combining Diogenes chemiluminescence assay with electron paramagnetic resonance. Furthermore, we determine the dose–response relationship and the threshold dose for •O2 – production. This work aims to provide a quantitative, comparative assessment of the macrophage superoxide release induced by environmentally relevant PM, thereby linking the PM source and dose to a key pro-inflammatory pathway.

2. Methods

2.1. PM Generation and Collection

Biomass burning PM and plastic combustion PM were generated from flaming combustion within a Thermolyne 21135 tube furnace described in our previous studies , and briefly summarized below. The furnace was operated at 600 °C under a pressure of 750 Torr. High-density polyethylene (HDPE), low-density polyethylene (LDPE), poly­(ethylene terephthalate) (PET), polystyrene (PS), and poly­(vinyl chloride) (PVC) burning PM were chosen because they are common plastic polymer types. A total of 200 mg of each plastic was placed in a ceramic crucible and positioned within the quartz tube. Synthetic air was flushed through the tube at 1 L min–1 for 3–5 min, and the combustion smoke was collected. Biomass burning PM was generated from pine needles using flaming combustion. Naphthalene, limonene, and toluene SOA particles were generated using an oxidative flow reactor (OFR), as described in Wei et al. (2021). The VOC precursors were carried into the OFR in a humidified airflow (250–1000 ccm), where they were oxidized by •OH and O3.

All PM samples were collected on PTFE fluoropore filters (0.2 μm pore size, FGLP04700, Millipore). Fresh PTFE filters were used as filter blanks. The sample mass was determined by weighing the filters before and after collection, with masses ranging from 0.5 to 13.1 mg. For each sample type, one or two independent filter samples were taken and cellular •O2 – analysis was performed in triplicate. Samples were stored for up to 6 or 14 months at −20 °C until analysis for cellular •O2 – production and cytotoxicity, respectively. During this storage period, some of the reactive organic components might have decayed. Therefore, our interpretation focuses on the effects of comparatively stable components.

2.2. Cell Culturing and Sample Preparation

RAW 264.7 macrophage cells (ATCC TIB-71) were cultured and passaged in a complete medium consisting of Dulbecco’s modified Eagle medium (DMEM, GIBCO), 10% fetal bovine serum, and 1% penicillin. For cellular •O2 – measurement, cells were seeded into 96-well plates at a density of 4 × 104 cells mL–1 per well and incubated in 5% CO2 for 2–3 h to allow adherence to the bottom of the well plates. PM samples were extracted from filters by vortex dissolution in incomplete cell media (DMEM only) for 7 min. The extracts were kept at room temperature for 0.5–1.5 h before cell exposure. PM concentrations were determined by weighing the filter before and after extraction, and the extracts were serially diluted to the desired concentrations using an incomplete medium.

2.3. Cellular Superoxide Measurement

A Diogenes chemiluminescence assay (National Diagnostics), in combination with a continuous-wave electron paramagnetic resonance (Bruker, Germany), was used to quantify cellular •O2 – production based on the procedure described by Fang et al. (2022). In brief, after cell incubation, the complete medium was replaced by 50 μL of the Diogenes probe. An incomplete medium was first added, and then 15 μL of 1250 U mL–1 superoxide dismutase (SOD) if applicable and last 50 μL of sample extract were added in sequence to each well to make a total volume of 200 μL. SOD, prepared in phosphate-buffered saline, is a superoxide scavenger and was added only to the control group to correct for any luminescence not generated from •O2 –. Once the sample extract was added, the exposure was considered to begin. Luminescence in Relative Light Unit (RLU) was monitored every 3 min for 4 h using a microplate reader (GloMax, Promega Corp.) with the chemiluminescence mode.

The calibration curve in work by Fang et al. (2022) was used to convert RLU to the cellular •O2 – production rate (μM min–1). The •O2 – production rates from the samples were corrected by those from the SOD, acellular chemical, and media or filter blank controls. The total •O2 – production was calculated by integrating the rate over the 4-h exposure period. Acellular chemical controls, which contained no cells but were exposed to the test samples, were processed on the same day as the cellular experiments. The media control refers to cellular production from cells not exposed to the PM sample, and an incomplete medium was used in place of sample extract. If more than one filter was used for sample analysis, the results were shown individually for different dose amounts or averaged for the same dose.

2.4. Cell Cytotoxicity

Cell cytotoxicity was measured using the CellTox Green Cytotoxicity Assay (Promega Corporation) for naphthalene SOA and PVC burning PM. Other PM types were not analyzed due to sample limitations. The protocol was adopted from work by Fang et al. (2022). Cells were plated at a cell density of 6 × 103 cells mL–1 and exposed to sample extracts for 4 h. Controls included filter blanks, a media blank with cells, and chemical background of reagent without cells. Lysis was added to a media control sample to determine maximum fluorescence. After the 4-h exposure, the assay reagent was added, plates were incubated for 15 min, and fluorescence was measured with the GloMax microplate reader with the fluorescence mode at an excitation wavelength of 475 nm and an emission wavelength of 520–530 nm.

3. Results

3.1. Time Profile

Parts a–i of Figure show the time profiles of the •O2 – production rate by macrophage cells exposed to various types of PM including limonene SOA (a), naphthalene SOA (b), toluene SOA (c), biomass burning PM (d), HDPE burning PM (e), LDPE burning PM (f), PVC burning PM (g), PET burning PM (h), and PS burning PM (i). The cellular •O2 – production is shown in gray scale with different doses, with black representing the highest exposure dose. The cellular production is corrected by acellular chemical controls (processed without cells but with full exposure to test samples), which are not shown because they yielded negligible •O2 –, indicating that chemical (noncellular) •O2 – production from all PM types was insignificant following extraction (0.5–1 h). This is consistent with work by Fang et al. (2022), who observed much higher cellular production than chemical production for isoprene SOA and 9,10-phenanthrenequinone.

1.

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•O2 – production rate (μM min–1) from macrophage cells exposed to limonene SOA (a), naphthalene SOA (b), toluene SOA (c), biomass burning PM (d), HDPE burning PM (e), LDPE burning PM (f), PVC burning PM (g), PET burning PM (h), and PS burning PM (i). All production rates have been media- or filter-blank-corrected.

As shown in Figure , the distinct time evolution plots indicate that the cellular response changes with each type of PM exposure. Some PM types enhance cellular •O2 – production at all tested doses, whereas other PM types have production maxima at specific doses. Limonene SOA (Figure a) induces a sharp increase in the •O2 – production rate for doses of 400–1600 μg mL–1. Naphthalene SOA (Figure b) exposure shows an increase in the •O2 – production rate throughout the entire exposure time for doses of 0.025–5 μg mL–1. A decrease of •O2 – is observed for doses of >25 μg mL–1. A previous study has shown that black carbon coated with 1,4-naphthoquinone, a product of naphthalene oxidation, showed significant ROS enhancement and cell viability reductions at doses of >50 μg mL–1. This suggests that the observed reduction of •O2 – production at doses of >25 μg mL–1 could be due to cell death. Toluene SOA (Figure c) shows a slight increase in the •O2 – production rate for cells exposed to 0.25–2 μg mL–1. Biomass burning PM (Figure d) induces a slight increase in the •O2 – production rate at doses of 2–60 μg mL–1. HDPE burning PM (Figure e) shows very limited enhancement. LDPE burning PM shows slight •O2 – production rate enhancement for lower doses (<10 μg mL–1). PVC, PET, and PS burning PM show sharp •O2 – production enhancements at specific doses. PVC and PET burning PM show a clearly enhanced •O2 – production rate for doses of 50–100 μg mL–1 (Figure g) and 100–500 μg mL–1 (Figure h), respectively. PS burning PM (Figure i), on the other hand, shows production enhancement at doses primarily under 25 μg mL–1.

3.2. Dose–Response Relationships

Figure shows the dose–response relationship for total cellular •O2 – production induced by each type of PM. Total •O2 – production was obtained by integrating the area under the curve from the kinetics of •O2 – release over the exposure time (Figure ). In general, for all PM types, total •O2 – production by macrophages increases with the dose and then decreases after a certain dose. This pattern is consistent with a respiratory burst response and with the trends reported for isoprene SOA and 9,10-phenanthrenequinone by Fang et al. (2022). The peak production, representing the maximum total •O2 – output, is a useful indicator of the potential maximum oxidative stress induced by a given PM.

2.

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Total •O2 – production (μM) from macrophage cells exposed to various doses of limonene SOA (a), naphthalene SOA (b), toluene SOA (c), biomass burning PM (d), HDPE burning PM (e), LDPE burning PM (f), PVC burning PM (g), PET burning PM (h), and PS burning PM (i). Error bars represent the standard deviations from triplicate measurements.

Among all tested PM, limonene SOA elicits the highest peak •O2 – production (∼32 μM). For comparison, Fang et al. (2022) showed that 9,10-phenanthrenequinone produced a higher peak of ∼100 μM, but this occurred at a much lower dose (∼0.87 μg mL–1) compared to limonene SOA reaching its peak at a substantially higher dose (∼700 μg mL–1). Fang et al. (2022) also found that isoprene SOA induced a peak production of ∼17 μM at a dose of 260 μg mL–1, while at a comparable dose, limonene SOA does not induce significant cellular •O2 – production. Exposure to naphthalene SOA (Figure b) results in a peak production of ∼6 μM at ∼0.03 μg mL–1, beyond which the response declines with increasing doses. Above 10 μg mL–1, •O2 – production was suppressed. Cytotoxicity analysis reveals elevated cell death at approximately this same dose (10 μg mL–1, Figure S1a), suggesting that the observed •O2 – suppression by naphthalene SOA may be attributable to cell death. Similarly, we observe an inverted V-shape distribution for toluene SOA (Figure c), which induces a peak production of 7 μM at a dose of 0.25 μg mL–1 and a suppression of •O2 – at doses higher than 3 μg mL–1.

Biomass burning PM exposure induces slight •O2 – production (<2.5 μM) between 0.01 and 20 μg mL–1, and a clear •O2 – suppression trend is observed beyond 100 μg mL–1 (Figure d). The plastic burning PM types exhibit distinct dose–response patterns. HDPE burning PM follows a similar inverted V shape, peaking at 10 μg mL–1 with a •O2 – production of <10 μM and showing a suppression beyond 90 μg mL–1 (Figure e). In contrast, LDPE burning PM does not display a clear •O2 – production peak; •O2 – production increases by ∼5 μM across all doses up to 500 μg mL–1, above which •O2 – suppression occurs (Figure f). PVC and PET burning PM (Figure g,h) are the only plastic burning PM to produce a sharp increase of •O2 – production. PVC burning PM has a minor peak (2.5 μM) at 0.1 μg mL–1 and a maximum of ∼7 μM at 50 μg mL–1. •O2 – production declines sharply beyond 80 μg mL–1, consistent with the observed increased cytotoxicity at comparable doses (Figure S1b). PET burning PM induces the highest •O2 – production among all plastics, reaching 12 μM at 100 μg mL–1, second only to limonene SOA overall. Lastly, PS burning PM shows a slight decreasing trend up to 10 μg mL–1, followed by a steeper decline at higher doses.

For naphthalene SOA and PVC burning PM, cytotoxicity measurements reveal elevated cell death at doses corresponding to the decline in •O2 – production (Figure S1), suggesting that cell death contributes to the observed suppression at high doses. We acknowledge that cytotoxicity data were not available for other PM types due to sample mass limitations, which restricts the full interpretation of their dose–response curves. For limonene SOA, toluene SOA, biomass burning PM, and other plastic burning PM, the decrease in •O2 – production at high doses could also arise from noncytotoxic mechanisms such as impaired phagocytic activity due to particle overload or direct inhibition of the enzyme by certain PM components. , Without viability data, we cannot distinguish between these possibilities and cytotoxicity. This represents a clear limitation of the current study, and future work incorporating comprehensive viability assays across all PM types will be necessary to fully resolve the mechanisms underlying the high-dose suppression.

3.3. Activation Thresholds

We investigated the minimum dose needed to activate •O2 – production, which we refer to as the activation threshold. The dose–response relationship observed in PM exposure to macrophages is nonlinear; thus, the relative toxicity cannot be compared at a single dose. The activation threshold therefore provides a dose metric for comparing the oxidative stress potential of different PM types. It distinguishes PM that requires high accumulation to trigger a response from PM that activates macrophages at trace concentrations. In this work, we determined the activation threshold as the dose at which superoxide production exceeds 5.7 μM based on 3 times the standard deviation of negative controls. Figure shows activation threshold vs •O2 – production at its peak dose for all PM tested. For SOA samples, limonene SOA has the highest activation threshold of 300 μg mL–1, which is similar to the threshold of isoprene SOA. Naphthalene SOA and toluene SOA have much smaller activation thresholds of 0.02 and 0.1 μg mL–1, respectively. Biomass burning PM does not show a clear activation threshold. The activation thresholds for PS, LDPE, and HDPE burning PM are ∼0.01 μg mL–1, while those for PVC and PET burning PM are higher, in the range of 50–100 μg mL–1.

3.

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Activation threshold (μg mL–1) plotted against the observed maximum superoxide production (μM) from macrophages exposed to various types of PM. *Isoprene SOA data from work by Fang et al. (2022). The error bars represent the standard deviation of triplicate measurements.

Interestingly, a lower activation threshold corresponds to lower peak •O2 – production and a higher activation threshold induces higher peak •O2 – production. Naphthalene SOA in previous studies was shown to have a higher oxidative potential than biogenic SOA. − Note that Fang et al. (2022) found that the activation threshold for another quinone, 9,10-phenanthrenequinone (PQN), was very low at 0.03 μg mL–1 with a very high •O2 – production of ∼100 μM (these data are not plotted because they are outside of the y-axis scale), an order of magnitude higher than any PM in this study. At the lowest dose tested, •O2 – production for PQN was already ∼20 μM. We believe there may be a stark difference observed for PQN because it is a pure quinone solution, whereas naphthalene SOA contains only 0.5–1.5% of napthoquinones, and other components such as organic peroxides, environmentally persistent free radicals (EPFRs), and unsaturated carbonyls compounds may contribute to the reactivity. − We also note that PQN and naphthoquinones differ in molecular structure, one-electron reduction potential, and biological activity; − therefore, the comparison with PQN is intended to be illustrative and does not represent a direct quantitative potency comparison.

The absence of a clear threshold for biomass burning PM is surprising given its frequently reported high toxicity in the literature. We have several possible explanations. First, prolonged storage at −20 °C may have led to the partial degradation of labile reactive components. Second, biomass burning PM may exert its toxicity through pathways other than the respiratory burst. Recent studies have shown that wildfire PM exposure can impair macrophage phagocytosis, and biofuel smoke-derived PM2.5 has been found to activate the NLRP3 inflammasome, leading to IL-1β and IL-18 release, an inflammatory mechanism distinct from NADPH oxidase-mediated superoxide production. Third, the chemical composition and toxicity of biomass burning PM vary significantly with the fuel type and combustion conditions. Our pine needle flaming combustion PM may differ in redox-active profiles from other biomass sources reported in the literature. Fourth, the lack of cytotoxicity data for biomass burning PM in our study limits our ability to determine whether the absence of a response reflects the absence of toxicity or functional suppression of the macrophage activity at the doses tested. These considerations highlight the need for future studies with freshly collected samples, complementary toxicity end points, and comprehensive compositional characterization.

4. Discussion

We found that the response of macrophages to release •O2 – via a respiratory burst upon exposure to PM is not a simple function of mass concentration but is instead governed by the chemical composition and dose of PM exposure. We observed that different PM types elicit four distinct patterns of •O2 – release: (1) a sharp, high-magnitude peak at a relatively high dose (e.g., limonene SOA and PET and PVC burning PM), (2) a broad, low-magnitude enhancement across a wide dose range (e.g., toluene SOA), and (3) a very low-threshold and low-magnitude enhancement (e.g., HDPE, PS, and LDPE burning PM), and (4) no clear threshold or obvious •O2 – peak (e.g., biomass burning PM). These patterns likely reflect differences in the underlying PM components that trigger or suppress the phagocytic oxidative burst. Water-soluble components that are chemically labile such as organic hydroperoxides can rapidly generate ROS upon dissolution, , potentially explaining the early and sharp •O2 – burst observed for limonene SOA, which contains substantial amounts of peroxides. However, not all hydrophilic PM elicit such a sharp response. Toluene SOA contains substantial amounts of hydrophilic compounds, yet it exhibits a low-magnitude, broad-range response without a sharp peak. This suggests that the chemical identity of hydrophilic species matters beyond solubility. In contrast, hydrophobic constituents (e.g., polycyclic aromatic hydrocarbons, quinones, and EPFRs) are more likely to partition into lipid membranes or require phagosomal internalization and sustained intracellular release to exert redox activity. The distinct dose–response patterns of plastic burning PM are consistent with their chemical composition characterized in our recent study. PVC and PET burning PM, which exhibited high-threshold, high-magnitude superoxide responses, contain high EPFR concentrations and high hypochlorous acid (HOCl) yields, whereas PS burning PM, which activated macrophages at very low doses, has the highest total PAH content and high OP-DTT. LDPE and HDPE burning PM, which showed weak or low-threshold responses, had relatively low metal and PAH contents and, for HDPE, low EPFR and OP-DTT values. These observations suggest that the activation threshold and maximum superoxide yield of plastic burning PM are influenced by the balance of redox-active organics, metals, EPFRs, and chlorinating species rather than by PM mass alone.

The high-threshold, high-magnitude response observed for limonene SOA and certain plastic-derived PMs (PET and PVC) suggests that these PMs require substantial accumulation to elicit a response. From a toxicological perspective, this pattern suggests the presence of a biological buffer below which the cells do not perceive the PM as a threat sufficient to trigger ROS release. In environmental contexts, such PM types may pose a negligible oxidative stress risk at typical ambient concentrations but could become acutely hazardous during episodic high-exposure events (e.g., near-source emissions). Conversely, a low-threshold, low-magnitude response means that macrophages initiate ROS production at very low doses, but the maximum response is modest. This pattern implies that even trace amounts of these PMs can activate cellular ROS generation, potentially contributing to chronic oxidative stress over prolonged exposure periods, a scenario more relevant to urban background air pollution. The biological consequence may be cumulative damage rather than acute cytotoxicity. Therefore, we propose that the assessment of PM-induced oxidative stress, especially for complex mixtures like SOA or combustion-derived PM, should not rely solely on a single time point or dose. Instead, it should incorporate a wide, dense dose range, time-revolved ROS measurement and reporting of both the activation threshold and maximum yield.

Additionally, it would be interesting to investigate the relationship of the activation threshold with the PM oxidative potential (OP), which receives growing attention as an indicator of PM toxicity. , Naphthalene SOA has been shown to have a much higher OP, as characterized by the DTT assay, compared to biogenic SOA. − This is qualitatively consistent with a smaller activation threshold for naphthalene SOA. For quantification of OP, there are multiple assays by measuring the decay rates of DTT, ascorbic acid, and glutathione and by measuring the formation rates of OH radicals. Comparison of the activation threshold for respiratory burst, cellular viability, and multiple OP assays may provide useful insights for characterization of the PM toxicity. We acknowledge that prolonged storage at −20 °C might have altered the chemical composition of PM samples and future studies employing immediate analysis or −80 °C storage would be valuable to further minimize storage artifacts.

Supplementary Material

ao6c06562_si_001.pdf (149.1KB, pdf)

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.6c06562.

  • Cell cytotoxicity for macrophages exposed to naphthalene SOA and PVC PM (Figure S1) (PDF)

We are thankful for our funding source, the U.S. National Science Foundation (CHE-2203419), and support from the Guangdong Provincial Project (2023QN10L005) and the Guangzhou–HKUST­(GZ) Joint Funding Program (No. 2025A03J3948).

The authors declare no competing financial interest.

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