A semi-automated system for quantifying the oxidative potential of ambient particles in aqueous extracts using the dithiothreitol (DTT) assay: results from the Southeastern Center for Air Pollution and Epidemiology (SCAPE)

Abstract

Abstract. A variety of methods are used to measure the capability of particulate matter (PM) to catalytically generate reactive oxygen species (ROS) in vivo, also defined as the aerosol oxidative potential. A widely used measure of aerosol oxidative potential is the dithiothreitol (DTT) assay, which monitors the depletion of DTT (a surrogate for cellular antioxidants) as catalyzed by the redox-active species in PM. However, a major constraint in the routine use of the DTT assay for integrating it with large-scale health studies is its labor-intensive and time-consuming protocol. To specifically address this concern, we have developed a semi-automated system for quantifying the oxidative potential of aerosol liquid extracts using the DTT assay. The system, capable of unattended analysis at one sample per hour, has a high analytical precision (coefficient of variation of 15 % for positive control, 4 % for ambient samples) and reasonably low limit of detection (0.31 nmol min −1 ). Comparison of the automated approach with the manual method conducted on ambient samples yielded good agreement (slope = 1.08 ± 0.12, r 2 = 0.92, N = 9). The system was utilized for the Southeastern Center for Air Pollution & Epidemiology (SCAPE) to generate an extensive data set on DTT activity of ambient particles collected from contrasting environments (urban, roadside, and rural) in the southeastern US. We find that water-soluble PM 2.5 DTT activity on a per-air-volume basis was spatially uniform and often well correlated with PM 2.5 mass (r = 0.49 to 0.88), suggesting regional sources contributing to the PM oxidative potential in the southeastern US. The correlation may also suggest a mechanistic explanation (oxidative stress) for observed PM 2.5 mass-health associations. The heterogeneity in the intrinsic DTT activity (per-PM-mass basis) across seasons indicates variability in the DTT activity associated with aerosols from sources that vary with season. Although developed for the DTT assay, the instrument can also be used to determine oxidative potential with other acellular assays.

On the Redox Activity of Urban Aerosol Particles: Implications for Size Distribution and Relationships with Organic Aerosol Components

“…Higher wintertime DTT activity has been frequently reported [9,24,26,27,36,53], on the contrary, other studies found higher activity in summer as compared to winter [31,44,47].…”

Development, characterization and first deployment of an improved online reactive oxygen species analyzer

“…One of the important pathways leading to deleterious impacts on health is believed to be induced oxidative stress by the generation of reactive oxygen species (ROS), through the interaction of particulate matter with the human lung (Donaldson et al, 2002). Reactive oxygen species denote chemically reactive molecules containing oxygen (e.g., radicals, oxygen ions and peroxides including the OH radical, the O − 2 radical, H 2 O 2 or organic peroxides) (Fuller et al, 2014;Sagai et al, 1993).…”

“…Currently, many acellular assays exist for the determination of ROS quantities in particles, including dithiothreitol (DTT) (Fang et al, 2015) and 2 ,7 -dichlorofluorescin (DCFH) (Fuller et al, 2014;King and Weber, 2013;Wang et al, 2011). The DCFH assay is one of the most commonly used assays today.…”

Inflammatory responses to secondary organic aerosols (SOA) generated from biogenic and anthropogenic precursors

“…Various assays have been developed to study PM-induced oxidant production, including cell-free chemical assays that measure the oxidative potential of PM samples (Kumagai et al, 2002;Cho et al, 2005;Fang et al, 2015b) as well as cellular assays that measure intracellular ROS/RNS produced as a result of PM exposure (Landreman et al, 2008;Tuet et al, 2016). Cell-free assays simulate biologically relevant redox (reduction-oxidation) reactions using an antioxidant species (e.g., dithiothreitol, DTT; ascorbic acid, AA).…”

“…5;Tuet et al, 2016). These ambient samples were analyzed using the same methods for determining oxidative potential (DTT assay; Cho et al, 2005;Fang et al, 2015b) and ROS/RNS production (cellular carboxy-H 2 DCFDA assay; Tuet et al, 2016). Furthermore, the same extraction protocol (water-soluble extract) was followed in both studies (Tuet et al, 2016).…”