Review of acellular assays of ambient particulate matter oxidative

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Review of acellular assays of ambient particulate matter oxidative potential: methods and relationships with composition, sources, and health effects Josephine Taylor Bates, Ting Fang, Vishal Verma, Linghan Zeng, Rodney J. Weber, Paige E Tolbert, Joseph Abrams, Stefanie Ebelt Sarnat, Mitchel Klein, James A Mulholland, and Armistead G. Russell Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b03430 • Publication Date (Web): 04 Mar 2019 Downloaded from http://pubs.acs.org on March 4, 2019

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Review of acellular assays of ambient particulate matter oxidative potential: methods and

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relationships with composition, sources, and health effects

3

Josephine Bates1, Ting Fang2, Vishal Verma3, Linghan Zeng4, Rodney Weber4, Paige Tolbert5,

4

Joseph Abrams6, Stefanie Sarnat5, Mitchel Klein5, James Mulholland1, Armistead Russell1*

5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28

1

Civil and Environmental Engineering, Georgia Institute of Technology, Atlanta, GA, USA

[email protected], [email protected], [email protected] 2

Department of Chemistry, University of California Irvine, Irvine, CA

[email protected] 3

Civil and Environmental Engineering, University of Illinois at Urbana-Champaign, Champaign, IL, USA

[email protected] 4

Earth and Atmospheric Sciences, Georgia Institute of Technology, Atlanta, GA, USA

[email protected], [email protected] 5

Rollins School of Public Health, Emory University, Atlanta, GA, USA

[email protected], [email protected], [email protected], 6

Center for Disease Control and Prevention, Atlanta, GA, USA

[email protected]

*corresponding author [email protected] 404.894.3079 311 Ferst Dr NW Ford Environmental Science & Technology Building 3210 Atlanta, GA 30332

ABSTRACT Acellular OP Assays

PM2.5

compositional impacts on OP?

GSH DTT AA ESR HPLC

oxidative stress-related health impacts?

Asthma COPD IHD

29 30

Oxidative stress is a potential mechanism of action for particulate matter (PM) toxicity and

31

can occur when the body’s antioxidant capacity cannot counteract or detoxify harmful effects of

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reactive oxygen species (ROS) due to an excess presence of ROS. ROS are introduced to the

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body via inhalation of PM with these species present on and/or within the particles (particle-

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bound ROS) and/or through catalytic generation of ROS in vivo after inhaling redox-active PM

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species (oxidative potential, OP). The recent development of acellular OP measurement

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techniques has led to a surge in research across the globe. In this review, particle-bound ROS

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techniques are discussed briefly while OP measurements are the focus due to an increasing

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number of epidemiologic studies using OP measurements showing associations with adverse

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health effects in some studies. The most common OP measurement techniques, including the

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dithiothreitol assay, glutathione assay, and ascorbic acid assay, are discussed along with

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evidence for utility of OP measurements in epidemiologic studies and PM characteristics that

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drive different responses between assay types (such as species composition, emission source, and

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photochemistry). Overall, most OP assays respond to metals like copper than can be found in

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emission sources like vehicles. Some OP assays respond to organics, especially photochemically

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aged organics, from sources like biomass burning. Select OP measurements have significant

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associations with certain cardiorespiratory endpoints, such as asthma, congestive heart disease,

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and lung cancer. In fact, multiple studies have found that exposure to OP measured using the

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dithiothreitol and glutathione assays drives higher risk ratios for certain cardiorespiratory

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outcomes than PM mass, suggesting OP measurements may be integrating the health-relevant

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fraction of PM and will be useful tools for future health analyses. The compositional impacts,

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including species and emission sources, on OP could have serious implications for health-

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relevant PM exposure. Though more work is needed, OP assays show promise for health studies

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as they integrate the impacts of PM species and properties on catalytic redox reactions into one

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measurement, and current work highlights the importance of metals, organic carbon, vehicles,

55

and biomass burning emissions to PM exposures that could impact health.

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1. Introduction

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Extensive literature supports the association between airborne particulate matter (PM)

58

and adverse human health effects, especially cardiorespiratory endpoints 1-7. However, the

59

mechanisms of action for PM-related health effects are not completely understood. Growing

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evidence in DNA methylation observations, animal models, and human biomarker studies shows

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that PM exposure can induce oxidative stress in the body, offering one potential mechanism of

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PM toxicity 5, 6, 8-16. Oxidative stress occurs when the concentration of reactive oxygen species

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(ROS) are in excess of the body’s antioxidant capacity, leading to a redox state change in cells

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that, in turn, can initiate or exacerbate inflammation in the respiratory tract and cardiovascular

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systems, chemically alter DNA, proteins, and lipids, and lead to cell and tissue damage or death

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5, 6, 8, 13.

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making them highly reactive, and include species like hydrogen peroxide (H2O2), superoxide

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radical (O2-), and hydroxyl radical (•OH). These species can be introduced to the body via

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particulate matter inhalation with ROS directly bound to the particles (particle-bound ROS)

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and/or by catalytic generation of ROS in vivo via cellular redox reactions stimulated by specific

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inhaled PM components 10, 17. The catalytic generation of ROS by such inhaled components with

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simultaneous depletion of antioxidants is defined in this paper as “oxidative potential” (OP).

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The recent development of acellular assays for OP has led to a rapid rise in OP

ROS are any oxygen-containing molecules that have one or more unpaired electrons,

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measurements worldwide using varying methods with differing results. Acellular assays are

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currently faster and less resource intensive than cellular assays, allowing more rapid

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development of reasonably large data sets in different locations for use in source apportionment

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and health analyses. Common acellular OP assays include electron spin (or paramagnetic)

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resonance (OPESR), dithiothreitol assay (OPDTT), ascorbic acid assay (OPAA), and glutathione

79

assay (OPGSH). OPESR measures the generation of hydroxyl radicals via electron spin resonance

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while OPDTT, OPAA, and OPGSH measure the depletion rate of chemical proxies for cellular

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reductants (DTT) or antioxidants (AA, GSH) which is proportional to the generation rate of

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ROS. Particle-bound ROS measurements use fluorescent-based techniques to measure

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concentrations of specific ROS, usually the hydroxyl radical or hydrogen peroxide, on and/or

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within a PM sample. The development of particle-bound ROS measurements precedes the

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development of acellular OP assays, but recent literature has shown the relevance of OP to

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health. Studies using cellular assays or focusing on the impact of persistent free radicals (PFR)

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are outside the scope of this review and can provide additional information on the relationship

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between PM exposure and health not discussed here, especially with regards to biological

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mechanisms.

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Epidemiologic analyses using acellular assays have found that exposure to PM with high

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OP affects cardiorespiratory health 18-21. In fact, OP was more strongly associated with acute

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cardiac and respiratory endpoints than fine PM concentration in multiple studies, suggesting that

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OP may be a more relevant health metric than PM mass for certain outcomes of interest 18, 19, 22.

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OP measurements integrate multiple aspects of PM, including species composition, synergistic

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interactions between chemical species and emission source impacts, redox cycling by complex

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organics, and oxidative stress delivered by surfaces, making it a potentially advantageous health

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metric with shown associations with acute cardiorespiratory health.

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Species and source composition, photochemical aging, volatility, and pH affect OP and size distribution of OP measurements. Organic compounds can generate oxidative stress through

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redox cycling of quinone-based radicals and metals can directly support electron transport to

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generate ROS while diminishing antioxidant levels 23. In general, all OP methods are responsive

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to metals, though the types of metals that drive responses and the degrees of those responses vary

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by assay. For example, OPAA is more sensitive to iron and copper and OPDTT to copper and

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manganese. OPDTT is also sensitive to organic species, especially highly oxidized organics.

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Literature shows that PM related to vehicle emissions drives responses in all OP assays, most

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likely due to the high copper content in brake and tire wear, and biomass burning PM

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significantly contributes to OPDTT due to its large oxidized aromatic (e.g., quinone) fraction. Size

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distribution is a current topic of interest due to the ability of different size fractions to reach

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different parts of the lung, and OP size distribution can vary by OP assay. Understanding the

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current state of knowledge on acellular OP assays, along with their relationship to composition,

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sources, and PM physical properties, could guide future research investigating the epidemiologic

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relevance of OP assays and further our understanding on the relationship between PM and

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health.

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This review summarizes the current state of knowledge on the relationship between

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health endpoints and OP of ambient PM along with the sensitivities of various acellular OP

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assays to PM composition, emission sources, and size. Particle-bound ROS measurements are

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described in brief, but the focus is on OP measurement techniques due to the growing body of

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literature linking these assays to adverse health outcomes. Limited work is available assessing

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utility of particle-bound ROS measurements in health studies, so future work is needed before

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conclusions about the relevance or lack thereof of particle-bound ROS to health can be made;

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although one may expect the large levels of antioxidants in the lung lining fluid would minimize

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the effect of particle-bound ROS.

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2. Overview of Measurement Methods

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ROS associated with PM can exist on and/or within the particle itself or can be generated in vivo by constituents in the inhaled particles chemically interacting with fluids and cells in the

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body. There are various methods to measure these two different phenomena. Approaches that

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measure ROS that exist on and/or within particles, or “particle-bound ROS” techniques, typically

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report levels in units of concentration (e.g. nmol H2O2 equivalents m-3air). Techniques that

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quantify the catalytic generation of ROS under simulated biological conditions, or “OP”

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measurement techniques, use units of time rate-of-change of volume-based concentration (nmol

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or % depletion per min per m3 of air) or mass-based concentration (nmol or % depletion per min

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per μg of PM; sometimes referred to as intrinsic OP), depending on the assay. If particle-bound

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ROS are present on samples during the OP analyses, they will drive a response in the OP assay.

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However, significant sample storage times and short lifetimes of particle-bound ROS often limit

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their impact in OP assays. Volume-based concentration rates of OP are relevant for

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epidemiologic studies while mass-based concentration rates of OP are useful for comparing

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between OP observational studies. Both methods can provide information on the capacity of PM

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species and sources to potentially drive catalytic redox reactions in the body, which is

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hypothesized to lead to health impacts related to oxidative stress; however, the difference in

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measurement methods can make comparisons across studies difficult.

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2.1.1. Measuring Particle Bound ROS

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Measurements of ROS present within and/or on PM are typically measured using various

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fluorescence techniques adapted from intracellular ROS measurement techniques. These particle-

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bound ROS methods suspend particles in a reagent and measure the spectra of specific oxidation

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byproducts. Dichlorofluorescin (DCFH) is the most common probe used when quantifying

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particle-bound ROS 24, 25. This chemical is a non-fluorescent reagent that becomes fluorescent

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(DCF) when oxidized in the presence of ROS. DCFH is mixed with Horseradish Peroxidase

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(HRP) in a sodium phosphate buffer prior to analysis to catalyze reactions. Then the DCFH-HRP

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reagent is added to each sample filter. The solution is sonicated to extract ROS in the particles,

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and fluorogenic intensity of DCF is measured. Fluorescent intensity is converted to H2O2

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concentration using least squares regression with a H2O2 calibration assay to obtain the final

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particle-bound ROS measurement 24. Other particle-bound ROS methods can vary by reagent

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type and concentration, mixing time and method, and fluorescence measurement technology.

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Other common reagents include the 9-(1,1,3,3,tetramethylisoindolin-2-yloxyl-5-ethynyl)-10-

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(phenylethynyl)anthracene (BPEAnit) probe, aminophenyl fluorescamine (APF) probe, and the

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10-acetyl-3,7-dihydroxyphenoxazine (Amplex Red) probe 26-28.

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Molecular probes vary in their sensitivities to species. In general, the DCFH probe is the

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least specific method, reacting similarly with multiple ROS, including hydroxyl radical (•OH),

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H2O2, peroxyl radicals, and peroxynitrite. This is largely due to the ease of abstraction of the

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hydrogen atom located at the 9’ position of the DCFH molecule 27. APF reacts with •OH but not

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peroxynitrite, unlike the DCFH and Amplex Red probes, making it potentially useful for specific

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hydroxyl radical measurements 28.

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Artifacts can occur using these techniques during sonication. Increased sonication of

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BPEAnit in dimethyl sulfoxide (DMSO) and of DCFH-HRP in 10/90 ethanol/water are

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correlated with an increase in fluorescence, implying sonication results in the formation of ROS

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29.

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higher than the values obtained for PM samples using the same probe 29. Particle-bound ROS

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measurements are also sensitive to pH, reagent concentration, and extraction method but not

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incubation temperature 30. According to Huang et al., 30 optimized performance in stability,

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reliability, and operability for offline particle-bound ROS measurements occurs at a pH of a

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phosphate buffer at 7.2 with the DCFH-HRP reagent at a concentration of 10M DCFH + 0.5

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unit/ml HRP. Care should be taken in the laboratory setup and methods when using molecular

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probe techniques to measure particle-bound ROS.

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The amount of ROS generated upon sonication can be approximately two orders of magnitude

One limitation of off-line particle-bound ROS measurements is the very short lifetime of

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ROS, ranging from only a few minutes to a day or longer, so off-line measurement techniques

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with delays of hours to days may severely underestimate true particle-bound ROS concentrations

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31.

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On-line measurement technologies have been developed to measure particle-bound ROS, in

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particular, three using DCFH 31, 35-37. The main difference between the three on-line

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measurement techniques available is the particle collection method. One instrument collects PM

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in an aqueous HRP solution on a paper filter that then flows through Teflon tubing immersed in a

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water bath for 15 minutes 31. This instrument was further developed to be portable (Online

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Particle-bound ROS Instrument, OPROSI) for automated continuous field deployment over

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many hours or days 35. Another method uses a particle into liquid sampler (PILS), which allows

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particle collection at a high flow rate 27. The final method uses a mist chamber to collect particles

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for online particle-bound ROS measurement 36. Nevertheless, the growing epidemiologic

Specifically, H2O2 bound to particles has been shown to significantly decrease over time 32-34.

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evidence suggests OP measurement techniques are potentially more relevant than particle-bound

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ROS measurements, pushing future research towards OP measurements.

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2.1.2. Measuring Oxidative Potential (OP)

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OP is most commonly measured as the capacity of PM to oxidize target molecules over

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time. Common methods include OPDTT, OPAA, and OPGSH. Assays using antioxidants, including

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OPAA and OPGSH, can be performed in a chemical assay or in surrogate lung fluid (SLF).

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Currently, all OPGSH studies have been performed with SLF, but OPAA has been measured in

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chemical and SLF environments. To differentiate if AA depletion was measured in SLF or not,

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subscripts will be used throughout this review (e.g., OPAA versus OPAA SLF). Two other techniques

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measure the actual generation of ROS over time rather than the depletion of target molecules

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using electron spin resonance (OPESR) or high-performance liquid chromatography (HPLC) with

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fluorescent probes. Different OP assays capture different redox reactions that lead to the

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generation of different ROS species. For example, OPESR and HPLC techniques measure the

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production rate of specific ROS, including •OH and H2O2, while antioxidant depletion assays

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(OPAA and OPGSH) have not been shown to be correlated with the generation of any specific

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ROS. OPDTT is most correlated with O2- and H2O2 formation and is not correlated with •OH

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generation 38.

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OPESR measures the ability of PM to induce •OH formation in the presence of H2O2 by

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measuring the electron paramagnetic resonance signals of the spin trap 5,5-dimethyl-1-pyrroline-

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N-oxide byproduct DMPO-OH quartet as the average of total amplitudes expressed in arbitrary

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units (A.U.) 39, 40. HPLC techniques measure the concentration of reduced byproducts of

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chemicals reacting with •OH or H2O2, such as 2,3 dihydroxygenzoic acid (2,3 DHBA)—the

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hydroxyl radical adduct of salicylic acid, p-hydroxybenzoate (p-HBA)—forming from the

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reaction of •OH with benzoate, and parahydroxylphenyl acetic acid (POPHAA) dimer—a result

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of the oxidation of POPHAA by H2O2 41-43. Other techniques measure the fluorescence of probes,

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such as disodium terephthalate (TPT)—which reacts with •OH to form 2-hydroxyterephthalic

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acid (2 OHTA), over time 38, 44. Sample measurements are taken over time in order to determine

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a time-rate of change in •OH or H2O2 concentration. One limitation of the benzoate (BA) probe is

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that it binds with manganese, but •OH production from manganese is negligible so results should

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not be affected significantly 45.

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Other OP assays, including OPDTT and OPAA, measure the depletion of biologically

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relevant chemicals due to oxidation. OPDTT is a commonly used chemical assay that has been

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used in epidemiologic analyses 46. DTT is a surrogate for the cellular oxidant NADPH, which

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reduces oxygen to the superoxide anion (O2-). Overall, the rate of O2- generation by a PM sample

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is measured by the rate at which DTT is consumed, which is proportional to the concentration of

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redox-active species in the PM sample. Specifically, solvent-extracted (e.g., in water or

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methanol) PM samples are incubated with DTT and a potassium phosphate buffer for times

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varying from 15 – 90 minutes. Some studies add metal chelators, often Chelex 100 or in some

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studies, EDTA, to the mixture to eliminate certain effects of metals. A small aliquot is removed

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from the mixture at designated times and mixed with 1% w/v Trichloraoacetic acid (TCA) to

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quench DTT reactions. The aliquot is mixed with 0.5 mL 5,5-dithiobis-(2-nitrobenzoic acid)

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(DTNB) to form 2-nitro-5-mercaptobenzoic acid (TNB) by reacting with the residual DTT,

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which is then measured using a spectrometer. The DTT consumption rate, otherwise known as

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DTT activity, is determined from the linear slope of DTT consumption and is used as a measure

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of OP 46, 47. Work has been done to alter the DTT assay protocol for personal monitoring use, but

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most current studies focus on ambient or chamber PM samples 48. The chemical OPAA protocol is

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very similar to the OPDTT protocol 49. Semi-automated OPDTT and OPAA assays have been

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developed to reduce labor intensity 47, 50. Online systems for OPDTT have also been developed to

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avoid filter sampling of PM that may lose reactive species before analysis and provide better

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temporal resolution (between 3 minutes and 3 hours) 51, 52. One system couples a PILS with

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microfluidic-electrochemical detection of reduced DTT using a cobalt(II) phthalocyanine

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(CoPC) electrode 52, 53, while another uses a Liquid Spot Sampler 51. Nevertheless, offline

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systems have been more commonly used because online technoloogy is still relativley new.

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The chemiluminescent reductive acridinium triggering (CRAT) assay measures the

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interaction of reductants, such as DTT or GSH, and oxidants. These chemicals act as reducing

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agents leading to the formation of H2O2, which in turn reacts with acridinium ester after addition

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of a buffer and emits light that can be used to quantify rates of H2O2 production 54. CRAT is a

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relatively new technique that, to the best knowledge of the authors, has not yet been used in

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ambient PM studies.

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GSH OPAA SLF and OP SLF measure % depletion of antioxidants in SLF exposed to PM rather than

directly from PM samples exposed only to the depletion chemical of interest. SLF is composed

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of multiple antioxidants, which is more indicative of realistic lung conditions. However, the

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composition of SLF varies by study and affects OP measurements, with AA being the most

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critical component in SLF for reactions with metals, making comparison across studies difficult

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45.

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monitoring absorbance over time, and/or a DTNB-enzyme recycling assay to obtain %

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antioxidant depletion per unit time 20, 55-57.

The concentration of antioxidants in SLF is quantified at specific time intervals using HPLC,

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There are many OP assays, and even within each assay, protocols can vary, making

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results across studies difficult to compare. Differences in methods that can affect magnitudes of

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measurements within an OP assay include choices of PM extraction solvents, PM filter types,

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incubation times, and metal chelators. For example, OPAA, OPDTT, OPESR, and OPCRAT are lower

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for PM measured on quartz filters than Teflon filters 58. Further, the fraction of PM extracted

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(water-soluble or water-insoluble) can affect OP results. Methanol can extract hydrophilic

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species and hydrophobic organic species, resulting in higher OPDTT than water-soluble extracts

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58-60.

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type, etc.), it is critical examine which OP assays are relevant for epidemiologic analyses in order

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to focus research on optimizing methods for these assays and to further the understanding of the

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association between PM and health.

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3. OP Measurements in Epidemiologic Analyses

Due to the differences in methods (use of SLF or not, choice of extraction method, filter

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Health studies focusing on OP have used OP measurement data and modeling

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approaches, such as land-use regression (LUR) and source impact regressions, to extend OP

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estimates where and when measurements are not available 18, 20, 49. Some studies use oxidative

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burden (OB), which is calculated by multiplying PM2.5 concentration from multiple locations or

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times by measured mass-based OP at one location and one time, assuming the mass-based OP

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does not vary significantly in time or space in the study area during the study period; [(µgPM_other

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sites

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to be associated with cardiorespiratory outcomes (Table 1). In some cases, those associations

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have been stronger than the associations between the same health outcome and PM mass,

275

supporting the hypothesis that oxidative stress is a mechanism of PM toxicity (Table 1). Based

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on current literature, OPDTT and OBGSH SLF are the OP assays most relevant to health.

277 278

m-3 )* (% depletion µgPM_one site-1min-1)]. Both modelled and measured OP have been found

Fractional exhaled nitric oxide (FeNO), used as a measure of airway inflammation, has been used in the most OP epidemiologic studies and studied with the most OP assays (OPESR,

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DTT, OBGSH) with mixed results (Table 1). A human exposure study in the OPAA, OPAA SLF, OP SLF

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Netherlands found a significant association between OPESR, OPAA, and OPDTT and FeNO after 5

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hours of exposure (to our knowledge, this is the only study to find an association between OPAA

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and health effects) 21. Interestingly, in two-pollutant models using two different OP

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measurements, effects of OPDTT remained after adjustment for OPESR and OPAA and vice versa,

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suggesting that the PM component drivers of OPDTT and OPESR or OPAA may have independent

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effects on FeNO. The same was not true for OPESR and OPAA as they were too correlated to

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disentangle their effects. A longer follow-up study in the Netherlands using OP estimates from a

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LUR model did not find an association between FeNO and OPESR but did find an association

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between FeNO and OPDTT 20. OPDTT has also been associated with FeNO in healthy adults after 2

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hours of exposure and in schoolchildren with persistent asthma in southern California (FeNO

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increased 8.7-9.9% per Interquartile Range (IQR): 0.43 nmol min-1 m-3) with exposures lagged

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one and two days 21, 61. The association of FeNO with OPDTT in the schoolchildren study was

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nearly twice as strong as with other measures (macrophage ROS and traffic-related markers) per

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AA IQR 61. OBGSH SLF was also shown to have an effect on FeNO up to 3 days after exposure, but OB SLF

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was not 62. OPAA has been found to have no association with other adverse health endpoints in

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multiple studies, including epidemiologic analyses on all-cause, respiratory, and cardiovascular

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mortalities, cardiorespiratory emergency department visits, myocardial infarction, and lung

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cancer mortality 22, 49, 63, 64, suggesting that OPAA may have limited utility in future epidemiologic

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studies. OPESR has had mixed results in limited health studies, and more work is needed before a

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conclusion of epidemiologic relevance is reached.

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Table 1. Reported results of tested associations between OP assays and health endpoints.

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Associations that were reported to be stronger for OP than PM concentration in at least one study

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are marked with an *. No significant negative (protective) associations were reported by any study. Health Endpoint Respiratory Health

Assays with Positive Associations

Assays with Null Associations

(confidence interval does not include null)

(confidence interval does include null)

Assay

Reference

General

DTT*

19 20, 21, 61

FeNO

DTT * ESR * GSHSLF

21, 65 62

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Reference

AASLF ESR GSHSLF

9, 62 20 9

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AA *

Asthma/Wheeze

DTT *

Lung Cancer Mortality

GSHSLF *

All-cause Mortality

-

21

18, 19, 49

22

AA AASLF UASLF GSHSLF

49

AASLF

22

AASLF GSHSLF

64

66 66 66

64

Cardiovascular Health Microvascular Function

DTT

67

Myocardial Infarction

GSHSLF

63

AASLF

63

Congestive Heart Failure

DTT *

18, 49

AA

49

Ischemic Heart Disease

DTT *

19

AASLF UASLF GSHSLF AASLF GSHSLF

66

Chronic Obstructive Pulmonary Disease All-cause Mortality

-

66 66 64 64

304 305 306

OPDTT has been linked with various acute cardiorespiratory endpoints in multiple studies

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(Table 1). Markers of respiratory health, including forced expiratory volume, asthma, and

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rhinitis, were more associated with LUR-estimated OPDTT than PM2.5 concentrations in a 14-year

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study in the Netherlands 20. Another study utilized a 10-year data set of modelled OPDTT in

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Atlanta, GA estimated via a regression with emission source impacts and found an association

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between OPDTT exposure and asthma/wheeze and congestive heart failure emergency department

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(ED) visits. In fact, OPDTT remained associated with ED visits in both one- and two-pollutant

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models while the PM2.5 association became null in the two-pollutant model, suggesting that

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OPDTT can explain much of the PM impact on these two health endpoints 18. A similar, shorter

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term (196 days), epidemiologic study was conducted on OPDTT measurements in Atlanta, GA

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rather than modelled concentrations, and this study showed a similarly significant association

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between OPDTT exposure and asthma/wheezing and ischemic heart disease ED visits despite the

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small sample size 19. Finally, particle size may play a role in determining health effects

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associated with OPDTT exposure as a study in Los Angeles showed an inverse relationship

ACS Paragon Plus Environment

Environmental Science & Technology

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between microvascular function in elderly adults and 5-day average OPDTT of PM