Particulate matter (PM) with an aerodynamic diameter less than 2.5 µm (fine PM or PM _2.5), is associated with diverse health problems and chronic diseases, including asthma, chronic obstructive pulmonary disease (COPD), lung cancer, and coronary heart disease ( Delfino et al., 2005; Delfino et al., 2011; Dockery et al., 1993; Dominici et al., 2006; Kaufman et al., 2016; Kim et al., 2013; Landrigan et al., 2018; Shah et al., 2013). Findings of recent epidemiological studies extend chronic PM _2.5 exposure risk to Alzheimer’s disease and accelerated cognitive decline ( Cacciottolo et al., 2017; Chen et al., 2015; Chen et al., 2017). Corresponding rodent models show robust indicators of inflammatory and oxidative stress to PM _2.5 fractions in pathological responses of aorta ( Li et al., 2003), brain ( Cheng et al., 2016b; Levesque et al., 2011; MohanKumar et al., 2008; Morgan et al., 2011), and lung ( Zhang et al., 2012).

In addition to the epidemiological associations with chronic disease, we must also consider diurnal variations in airborne particulate matter chemistry that are not included in most long-term epidemiological studies. Diurnal variation in air pollution toxicity is suggested by diurnal variations in emergency department admissions for dementia ( Linares et al., 2017), ischemic stroke ( Han et al., 2016), and respiratory conditions ( Darrow et al., 2011). Although these admissions were more strongly associated with ozone than with PM _2.5 in all three of these studies, diurnal changes in PM _2.5 chemistry must also be considered as an influencing factor. Freshly emitted primary PM undergoes photochemical oxidation reactions over the course of the day, catalyzed by ultraviolet (UV) sunlight, which results in diverse oxidized organic and inorganic products (secondary PM) ( Forstner et al., 1997; Grosjean & Seinfeld, 1989), along with concomitant changes in PM toxicity. These diurnal changes in PM _2.5 composition and associated toxicity are relevant to and may inform future long-term epidemiological studies of primary and secondary particulate matter. While prior studies in the Los Angeles air basin have shown extensive diurnal variations in PM composition and size, the findings of PM oxidative activity have been inconsistent and differ between various assays of oxidative potential ( Saffari et al., 2015; Verma et al., 2009; Wang et al., 2013b).

The current study further examined diurnal variations in composition and oxidative potential of PM samples collected at the central Los Angeles site used in the three studies mentioned above. However, unlike these earlier studies, PM samples were collected by a direct aerosol-into-liquid collection method to provide time-integrated aqueous PM _2.5 slurries for both morning and afternoon periods. This technology allows for a more comprehensive analysis than the filterable (i.e. water extracted) particulate samples examined in our prior studies ( Morgan et al., 2011; Saffari et al., 2015; Verma et al., 2009; Woodward et al., 2017a).

Microglia were used for in vitro assays of oxidative and inflammatory responses to PM _2.5 exposures because of their increasingly recognized role in environmental neurotoxicology ( Krafft, 2015). Air pollution can induce premature microglial activation, as documented in rodent models ( Cheng et al., 2016a; Hanamsagar & Bilbo, 2017; Morgan et al., 2011) and as indicated for young adults living in the highly polluted Mexico City ( Calderon-Garciduenas et al., 2008; Calderon-Garciduenas et al., 2018). Microglia (BV-2) cell cultures were assayed for induction of nitric oxide (NO) and for proinflammatory gene mRNA responses of interleukins 6 and 1β (IL-6 & IL-1β), and monocyte chemoattractant protein 1 (MCP-1), also known as chemokine (C-C motif) ligand 2 (CCL2). These markers were chosen because of their in vivo and in vitro responses to ultrafine PM shown in prior studies ( Cheng et al., 2016b; Morgan et al., 2011; Woodward et al., 2017b).

We hypothesized that afternoon PM _2.5 (pm-PM _2.5), with its high proportion of secondary photochemical oxidation products, would have greater oxidative and proinflammatory activity than freshly emitted, primary PM collected during morning hours (am-PM _2.5).

Diurnal variations in urban PM _2.5 oxidative and proinflammatory activity showed consistent decreases from morning to afternoon sampling periods in two independent in vitro assays using the BV-2 microglia cell line. The collection of total PM _2.5 as an aqueous slurry was enabled by direct aerosol-into-liquid sampling that more efficiently captures water-insoluble components of ambient PM _2.5 than traditional filter-based sampling methods used in several prior studies (e.g. Saffari et al., 2015; Verma et al., 2009). These slurry samples are more representative of the full range of ambient PM components and their toxicities than filter-trapped and water eluted PM. Additionally, the results of the NO assay and the qPCR assay for inflammatory gene responses extend findings from the widely used dithiothreitol (DTT) and alveolar macrophage (dichlorodihydrofluorescein, DCFH) assays for oxidative potential, which can be confounded by oxidative recycling from transition metals ( Forman & Finch, 2018). Our findings, that primary PM _2.5 results in a greater oxidative and proinflammatory response than secondary PM _2.5, are contrary to expectations based on prior reports that secondary, photo-oxidized PM exhibits greater oxidative activity than primary PM.

Previous studies of diurnal variations in PM composition and oxidative activity have not been consistent and were limited in using only simple assays of oxidative potential (i.e. DTT and DCFH) on filter-captured PM. Relying solely on oxidative potential measures such as the DTT and DCFH assays provides us with only an imprecise measure of cellular oxidative and proinflammatory activity that lacks specificity. The current study improves on the experimental design of past studies by utilizing direct measures of acute oxidative stress and inflammation, including free radical production induced by PM as nitric oxide (NO) and cellular proinflammatory mRNA responses. Additionally, by using the direct aerosol-into-liquid method to collect aqueous slurries in our study, water-insoluble PM species were more efficiently captured, providing samples more representative of the full range of ambient PM components and their toxicities.

Further insight into the sources of particulate toxicity may be gleaned by the apportionment of redox properties to its water soluble and insoluble chemical components, including water-soluble and water-insoluble organic carbon (WSOC and WIOC, respectively). WSOC species are generally defined as hydrophilic, while WIOC are hydrophobic ( Turpin & Lim, 2001). Wang et al. (2013b) collected aqueous PM _2.5 slurries by a similar aerosol-into-liquid sampling method, and found that increased WIOC content in PM _2.5, relative to WSOC content, was highly correlated with redox activity on a per mass basis, indicating a greater intrinsic toxicity of WIOC as compared to WSOC. While this study was limited by its use of the DCFH assay, the greater oxidative potential associated with increased WIOC mass concentrations was attributed to organic compounds such as PAHs, as well as iron and other transition metals.

Our results indicate that morning PM _2.5, which contains a greater proportion of water-insoluble species, may be intrinsically more toxic and induce greater cellular oxidative stress, than afternoon PM _2.5 samples that contain a larger mass fraction of oxidized, water-soluble species that are products of photochemical reactions in the atmosphere ( Seinfeld & Pandis, 2016), including the inorganic secondary ions NO ₃ ^-, SO ₄ ^2-, NH ₄ ⁺, and Na ⁺. The mechanisms underlying the greater toxicity of primary, morning PM _2.5 may involve non-polar WIOC components, such as PAHs, being able to more easily permeate the hydrophobic lipid-bilayer of cell membranes to trigger the formation of intracellular oxidative species and induce proinflammatory cytokine formation via an acute oxidative stress response.

Primary, traffic-derived PM _2.5 also consists of greater concentrations of redox active and other toxic metals, as compared to the bulk of secondary PM _2.5, which consists largely of hydrophilic products of photochemical oxidation. The metals and trace elements we found to be more prevalent in the morning slurry sample included the heavy metals vanadium, chromium, nickel, and arsenic, which are emitted by vehicles both as fuel combustion products as well as remnants of motor oil degradation ( Geller et al., 2006), copper, which is associated with vehicular brake wear ( Garg et al., 2000; Sanders et al., 2003; Sternbeck et al., 2002), and zinc, which is primarily a product of tire deterioration ( Singh et al., 2002). Elevated levels of these metals in both collection periods correspond to vehicular emissions as the major source of primary particles in close proximity to the I-110 freeway. We believe the higher proportions of these metals and WIOC components in primary PM _2.5 dominant in the morning hours, as compared to photo-oxidized secondary PM _2.5 prevalent in the afternoon, are responsible for the diurnal variation in acute oxidative stress observed in the current study.

The data presented in this study demonstrate that urban PM _2.5 collected during the morning rush hour (6–9am), when primary, traffic-derived PM emissions are dominant, induces greater oxidative and proinflammatory responses in cells as compared to PM _2.5 collected in the afternoon (12–4pm), which contains a higher proportion of photo-oxidized, secondary PM products. Two in vitro assays of the cellular inflammatory response consistently demonstrated greater oxidative and proinflammatory activity due to primary (morning) PM _2.5 exposure. We attribute this effect to the greater transition metal and water-insoluble organic carbon (WIOC) content of primary PM _2.5, two classes of PM components that increase toxicity ( Cho et al., 2005; Hu et al., 2008; Li et al., 2009; Shirmohammadi et al., 2015; Tao et al., 2003; Zhang et al., 2008). Our study also improves upon previous research of diurnal variations in PM-induced oxidative stress by utilizing a unique aerosol-into-liquid PM collection system that more efficiently captures water insoluble components, thus providing complete aqueous PM samples more representative of ambient PM.

This research will ultimately help us gain a more complete understanding of the complex nature of particulate matter and how its composition and proinflammatory effects change over time due to photochemical aging in the atmosphere. The Southern California climate of Los Angeles with abundant sunshine, compounded with dense vehicular traffic, generates ubiquitous primary and secondary PM throughout the year. Identifying the health effects of these pollutants is critical as we strive to understand the underlying mechanisms of PM-induced oxidative stress, neuroinflammation and associated morbidity. Our findings may help in further elucidating the role of PM in the etiology, onset and development of widespread, chronic diseases that plague urban populations, including cancer, cardiac and respiratory distress, and neurodegenerative disorders such as Alzheimer’s disease.

The data referenced by this article are under copyright with the following copyright statement: Copyright: � 2018 Lovett C et al.

Data associated with the article are available under the terms of the Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication).

Dataset 1: The following raw data sets are provided as comma separated values (.csv) files: 10.5256/f1000research.14836.d203329 ( Lovett et al., 2018)

PM_Diurnal_Variation_NO_Fig1_DATA

PM_Diurnal_Variation_qPCR_Fig2_DATA

PM_Diurnal_Variation_TC_Fig3A_DATA

PM_Diurnal_Variation_Ions_Fig3B_DATA

PM_Diurnal_Variation_Metals_Fig3C_DATA