Cellular and Acellular Assays for Measuring Oxidative Stress Induced by Ambient and Laboratory-Generated Aerosols.

INTRODUCTION: Many studies have established associations between exposure to air pollution, or atmospheric particulate matter (PM), and adverse health effects. An increasing array of studies have suggested oxidative stress as a possible mechanism by which PM-induced health effects arise, and as a result, many chemical and cellular assays have been developed to study PM-induced oxidant production. Although significant progress has been made in recent years, there are still many gaps in this area of research that have not been addressed. Many prior studies have focused on the aerosol of primary origin (e.g., the aerosol emitted from combustion engines) although the aerosol formed from the oxidation of volatile species, secondary organic aerosol (SOA), has been shown to be the predominant type of aerosol even in urban areas. Current SOA health studies are limited in number, and as such, the health effects of SOA are poorly characterized. Also, there is a lack of perspective in terms of the relative toxicities of different SOA systems. Additionally, although chemical assays have identified some SOA constituents associated with adverse health endpoints, the applicability of these results to cellular responses has not been well established. SPECIFIC AIMS: The overall objective of this study was to better understand the oxidative properties of different types and components of PM mixtures (especially SOA) through systematic laboratory chamber experiments and ambient field studies. The study had four specific aims.1 To develop a cellular assay optimized for measuring reactive oxygen and nitrogen species (ROS/RNS) production resulting from PM exposure and to identify a robust parameter that could represent ROS/RNS levels for comparison with different endpoints.2 To identify ambient PM components associated with ROS/RNS production and evaluate whether results from chemical assays represented cellular responses in terms of ROS/RNS production.3 To investigate and provide perspective on the relative toxicities of SOA formed from common biogenic and anthropogenic precursors under different conditions (e.g., humidity, nitrogen oxides [NOx], and redox-active metals) and identify bulk aerosol properties associated with cellular responses.4 To investigate the effects of photochemical aging on aerosol toxicity. METHODS: Ambient PM samples were collected from urban and rural sites in the greater Atlanta area as part of the Southeastern Center for Air Pollution and Epidemiology (SCAPE) study between June 2012 and October 2013. The concentrations of water-soluble species (e.g., water-soluble organic carbon [WSOC], brown carbon [Br C], and metals) were characterized using a variety of instruments. Samples for this study were chosen to span the observed range of dithiothreitol (DTT) activities.Laboratory studies were conducted in the Georgia Tech Environmental Chamber (GTEC) facility in order to generate SOA under well-controlled photooxidation conditions. Precursors of biogenic origin (isoprene, α-pinene, and ß-caryophyllene) and anthropogenic origin (pentadecane, m-xylene, and naphthalene) were oxidized under various formation conditions (dry vs. humid, NOx, and ammonium sulfate vs. iron sulfate seed particles) to produce SOA of differing chemical composition and mass loading. For the naphthalene system, a series of experiments were conducted with different initial hydrocarbon concentrations to produce aerosols with various degree of oxidation. A suite of instruments was utilized to monitor gas- and particle-phase species. Bulk aerosol properties (e.g., O:C, H:C, and N:C ratios) were measured using a high-resolution time-of-flight aerosol mass spectrometer. Filter samples were collected for chemical oxidative potential and cellular measurements. For the naphthalene system, multiple filter samples were collected over the course of a single experiment to collect aerosols of different photochemical aging.For all filter samples, chemical oxidative potentials were determined for water-soluble extracts using a semiautomated DTT assay system. Murine alveolar macrophages and neonatal rat ventricular myocytes were also exposed to PM samples extracted in cell culture medium to investigate cellular responses. ROS/RNS production was detected using the intracellular ROS/RNS probe, carboxy-2',7'-dichlorodihydrofluorescein diacetate (carboxy-H2DCFA), whereas cellular metabolic activity was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Finally, cytokine production, that is, secreted levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), were measured post-exposure using an enzyme-linked immunosorbent assay (ELISA). To identify PM constituents associated with oxidative properties, linear regressions between oxidative properties (cellular responses or DTT activity) and aerosol composition (metals, elemental ratios, etc.) were evaluated using Pearson's correlation coefficient, where the significance was determined using multiple imputation and evaluated using a 95% confidence interval. RESULTS: We optimized several parameters for the ROS/RNS assay, including cell density (2 × 104 cells/well for macrophages and 3.33 × 104 cells/well for cardiomyocytes), probe concentration (10 µM), and sample incubation time (24 hours). Results from both ambient and laboratory-generated aerosols demonstrate that ROS/RNS production was highly dose-dependent and nonlinear with respect to PM dose. Of the dose-response metrics investigated in this study (maximum response, dose at which the response is 10% above the baseline [threshold], dose at which 50% of the response is attained [EC50], rate at which the maximum response is attained [Hill slope], and area under the dose-response curve [AUC]), we found that the AUC was the most robust parameter whose informativeness did not depend on dose range.A positive, significant correlation was observed between ROS/RNS production as represented by AUC and chemical oxidative potential as measured by DTT for ambient samples collected in summer. Conversely, a relatively constant AUC was observed for ambient samples collected in winter regardless of the corresponding DTT activity. We also identified several PM constituents (WSOC, BrC, iron, and titanium) that were significantly correlated with AUC for summer samples. The strong correlation between organic species and ROS/RNS production highlights a need to understand the contribution of organic aerosols to PM-induced health effects. No significant correlations were observed for other ROS/RNS metrics or PM constituents, and no spatial trends were observed.For laboratory-generated aerosol, precursor identity influenced oxidative potentials significantly, with isoprene and naphthalene SOA having the lowest and highest DTT activities, respectively. Both precursor identity and formation condition significantly influenced inflammatory responses induced by SOA exposure, and several response patterns were identified for SOA precursors whose photooxidation products share similar carbon-chain length and functionalities. The presence of iron sulfate seed particles did not have an apparent effect on oxidative potentials; however, a higher level of ROS/RNS production was observed for all SOA formed in the presence of iron sulfate compared with ammonium sulfate. We also identified a significant positive correlation between ROS/RNS production and average carbon oxidation state, a bulk aerosol property. It may therefore be possible to roughly estimate ROS/RNS production using this property, which is readily obtainable. This correlation may have significant implications as aerosols have an atmospheric lifetime of a week, during which average carbon oxidation state increases because of atmospheric photochemical aging. Our results suggest that aerosols might become more toxic as they age in the atmosphere. Finally, in the context of ambient samples, laboratory-generated SOA induced comparable or higher levels of ROS/RNS. Oxidative potentials for all laboratory SOA systems, with the exception of naphthalene (which was higher), were all comparable with oxidative potentials observed in ambient samples.

Prebiotic Phosphorylation of Uridine using Diamidophosphate in Aerosols.

One of the most challenging fundamental problems in establishing prebiotically plausible routes for phosphorylation reactions using phosphate is that they are thermodynamically unfavorable in aqueous conditions. Diamidophosphate (DAP), a potentially prebiotically relevant compound, was shown to phosphorylate nucleosides in aqueous medium, albeit at a very slow rate (days/weeks). Here, we demonstrate that performing these reactions within an aerosol environment, a suitable model for the early Earth ocean-air interface, yields higher reaction rates when compared to bulk solution, thus overcoming these rate limitations. As a proof-of-concept, we demonstrate the effective conversion (~6.5-10%) of uridine to uridine-2',3'-cyclophosphate in less than 1 h. These results suggest that aerosol environments are a possible scenario in which prebiotic phosphorylation could have occurred despite unfavorable rates in bulk solution.

Real-time methods for estimating organic component mass concentrations from aerosol mass spectrometer data.

We use results from positive matrix factorization (PMF) analysis of 15 urban aerosol mass spectrometer (AMS) data sets to derive simple methods for estimating major organic aerosol (OA) component concentrations in real time. PMF analysis extracts mass spectral (MS) profiles and mass concentrations for key OA components such as hydrocarbon-like OA (HOA), oxygenated OA (OOA), low-volatility OOA (LV-OOA), semivolatile OOA (SV-OOA), and biomass burning OA (BBOA). The variability in the component MS across all sites is characterized and used to derive standard profiles for real-time estimation of component concentrations. Two methods for obtaining first-order estimates of the HOA and OOA mass concentrations are evaluated. The first approach is the tracer m/z method, in which the HOA and OOA concentrations are estimated from m/z 57 and m/z 44 as follows: HOA ∼ 13.4 × (C(57) - 0.1 × C(44)) and OOA ∼ 6.6 × C(44), where C(i) is the equivalent mass concentration of tracer ion m/z i. The second approach uses a chemical mass balance (CMB) method in which standard HOA and OOA profiles are used as a priori information for calculating their mass concentrations. The HOA and OOA mass concentrations obtained from the first-order estimates are evaluated by comparing with the corresponding PMF results for each site. Both methods reproduce the HOA and OOA concentrations to within ∼30% of the results from detailed PMF analysis at most sites, with the CMB method being slightly better. For hybrid CMB methods, we find that fixing the LV-OOA spectrum and not constraining the other spectra produces the best results.

Evolution of organic aerosols in the atmosphere.

Organic aerosol (OA) particles affect climate forcing and human health, but their sources and evolution remain poorly characterized. We present a unifying model framework describing the atmospheric evolution of OA that is constrained by high-time-resolution measurements of its composition, volatility, and oxidation state. OA and OA precursor gases evolve by becoming increasingly oxidized, less volatile, and more hygroscopic, leading to the formation of oxygenated organic aerosol (OOA), with concentrations comparable to those of sulfate aerosol throughout the Northern Hemisphere. Our model framework captures the dynamic aging behavior observed in both the atmosphere and laboratory: It can serve as a basis for improving parameterizations in regional and global models.

Measurements of secondary organic aerosol from oxidation of cycloalkenes, terpenes, and m-xylene using an Aerodyne aerosol mass spectrometer.

The Aerodyne aerosol mass spectrometer (AMS) was used to characterize physical and chemical properties of secondary organic aerosol (SOA) formed during ozonolysis of cycloalkenes and biogenic hydrocarbons and photo-oxidation of m-xylene. Comparison of mass and volume distributions from the AMS and differential mobility analyzers yielded estimates of "effective" density of the SOA in the range of 0.64-1.45 g/cm3, depending on the particular system. Increased contribution of the fragment at m/z 44, C02+ ion fragment of oxygenated organics, and higher "delta" values, based on ion series analysis of the mass spectra, in nucleation experiments of cycloalkenes suggest greater contribution of more oxygenated molecules to the SOA as compared to those formed under seeded experiments. Dominant negative "delta" values of SOA formed during ozonolysis of biogenics indicates the presence of terpene derivative structures or cyclic or unsaturated oxygenated compounds in the SOA. Evidence of acid-catalyzed heterogeneous chemistry, characterized by greater contribution of higher molecular weight fragments to the SOA and corresponding changes in "delta" patterns, is observed in the ozonolysis of alpha-pinene. Mass spectra of SOA formed during photooxidation of m-xylene exhibit features consistent with the presence of furandione compounds and nitro organics. This study demonstrates that mixtures of SOA compounds produced from similar precursors result in broadly similar AMS mass spectra. Thus, fragmentation patterns observed for biogenic versus anthropogenic SOA may be useful in determining the sources of ambient SOA.

Organ-bath studies using the irreversible beta-adrenoreceptor antagonist bromoacetylalprenololmenthane (BAAM).

1. The effects of the irreversible beta-adrenoreceptor antagonist bromoacetylalprenololmenthane (BAAM) were studied in isolated cardiac and uterine preparations from guinea-pigs and rats and in guinea-pig ileal preparations. 2. In the presence of BAAM (0.1-10 microM) concentration-effect curves to (-)-isoprenaline were shifted to the right in a concentration-dependent manner in all cardiac and uterine tissues. Maximum responses to (-)-isoprenaline were unaffected by BAAM except in guinea-pig left atrial and in some guinea-pig uterine preparations; however, the reductions in the maximum responses were not concentration-dependent. The mean pKB values for BAAM in guinea-pig left atria, right atria, rat whole atria and rat uterus were 7.26, 7.24, 6.84 and 7.90 respectively. 3. In guinea-pig ileal preparations, BAAM (0.1-30 microM) relaxed contractions induced by K+, histamine and acetylcholine in a non-beta-adrenoreceptor-related manner since relaxant responses were unaffected by propranolol (0.5 microM). In other tissues higher concentrations of BAAM (30-100 microM) elicited atrial standstill and depressed K+-induced contractions in uterine smooth muscle. 4. Treatment of tissues with BAAM (10-100 microM) followed by extensive wash-out increased the EC50 values for (-)-isoprenaline 21- to 83-fold. The maximum response to the catecholamine was unaffected by BAAM except in guinea-pig left atrial preparations following treatment with 100 microM BAAM. At these concentrations BAAM markedly increased the effective refractory period. 5. Concentration-effect curves for the partial agonist, oxymethylene-isoprenaline (OM-ISO) were shifted to the right 12- to 355-fold after pretreatment of tissues with BAAM (10-30 microM) followed by wash-out. The maximum response to OM-ISO was unaltered in guinea-pig and rat uteri and was reduced to a similar degree as observed with (-)-isoprenaline in guinea-pig left atria. 6. In general, the non-selective beta-adrenoreceptor antagonist BAAM depressed maximum responses to beta-adrenoreceptor agonists only in cardiac preparations and at concentrations which elicited depressant activity. On the basis of the present study, BAAM does not appear to be a suitable irreversible beta-adrenoreceptor antagonist for use in organ bath experiments.