Rethinking Dithiothreitol-Based Particulate Matter Oxidative Potential

May 10, 2017 - We measured the rate of generation of reactive oxygen species (ROS) [hydroxyl radicals (•OH) and hydrogen peroxide (H2O2)] catalyzed ...
5 downloads 0 Views 1MB Size
Article pubs.acs.org/est

Rethinking Dithiothreitol-Based Particulate Matter Oxidative Potential: Measuring Dithiothreitol Consumption versus Reactive Oxygen Species Generation Qianshan Xiong, Haoran Yu, Runran Wang, Jinlai Wei, and Vishal Verma* Department of Civil and Environmental Engineering, University of Illinois at Urbana−Champaign, 205 North Mathews Avenue, Urbana, Illinois 61801, United States S Supporting Information *

ABSTRACT: We measured the rate of generation of reactive oxygen species (ROS) [hydroxyl radicals (•OH) and hydrogen peroxide (H2O2)] catalyzed by ambient particulate matter (PM) in the dithiothreitol (DTT) assay. To understand the mechanism of ROS generation, we tested several redox-active substances, such as 9,10-phenanthrenequinone (PQ), 5-hydroxy-1,4-naphthoquinone (5H-1,4NQ), 1,2-naphthoquinone (1,2-NQ), 1,4-naphthoquinone (1,4-NQ), copper(II), manganese(II), and iron (II and III). Both pure compounds and their mixtures show different patterns in DTT oxidation versus ROS generation. The quinones, known to oxidize DTT in the efficiency order of PQ > 5H-1,4NQ > 1,2-NQ > 1,4NQ, show a different efficiency order (5H-1,4NQ > 1,2-NQ ≈ PQ > 1,4-NQ) in the ROS generation. Cu(II), a dominant metal in DTT oxidation, contributes almost negligibly to the ROS generation. Fe is mostly inactive in DTT oxidation, but shows synergistic effect in •OH formation in the presence of other quinones (mixture/sum > 1.5). Ten ambient PM samples collected from an urban site were analyzed, and although DTT oxidation was significantly correlated with H2O2 generation (Pearson’s r = 0.91), no correlation was observed between DTT oxidation and •OH formation. Our results show that measuring both DTT consumption and ROS generation in the DTT assay is important to incorporate the synergistic contribution from different aerosol components and to provide a more inclusive picture of the ROS activity of ambient PM.

1. INTRODUCTION Both short-1,2 and long-term3,4 exposure to ambient particulate matter (PM) have been linked to adverse health end points in humans. The toxicity of ambient PM appears to be a multifaceted phenomenon, probably involving many active chemical constituents.5 There could be several mechanisms induced by these components either individually6 or synergistically,7 leading to the observable toxic effects of ambient PM, however, most of the recent studies seem to be now converging on the initiating step of the PM toxicity ladder, which is the generation of reactive oxygen species (ROS).8−13 The resultant oxidative stress causes disruptions in normal mechanisms of cellular signaling leading to many pathophysiological conditions in the body such as neurodegenerative14,15 and inflammatory16,17 diseases. Recognizing the importance of this ROS generation step, a variety of chemical probes were developed to measure the oxidative properties of ambient particles.18−23 These assays simulate the conditions of the cellular environment to the extent possible in a chemical system and measure the PM oxidative potential either through direct measurement of ROS (e.g., high-performance liquid chromatography,24 spin trapping or electron spin resonance spectroscopy,25,26 and fluorescencebased method) 27,28 or indirectly (i.e. rate of loss of antioxidants).19,29 Various particle components seem to © XXXX American Chemical Society

participate differentially in the reactions simulated by these assays, therefore yielding an inconsistent relationship between the chemical composition and measured assay response. For example, ascorbate assay has been shown to be responsive mostly to Cu30 (similar to citrate being responsive mostly to Fe),31 and both Fe and Cu seem to play important roles in glutathione oxidation.32 Among all the chemical assays developed so far, the dithiothreitol (DTT) assay has been found to be responsive with the largest pool of chemical components, which involves many aromatic hydrocarbons,33 in addition to two transition metals: Cu and Mn.34 In a previous study measuring both the DTT and ascorbate activities (i.e., DTT and ascorbate oxidation capabilities) of a large number of PM samples (N > 500) collected from the southeast United States,29 the authors found that while DTT activity was associated with three major emission sources (i.e., vehicular emissions, biomass burning, and secondary formation), only vehicular emissions (a major source of Cu) was associated with the response of ascorbate assay. Moreover, DTT activity has been shown to be correlated Received: Revised: Accepted: Published: A

March 9, 2017 May 4, 2017 May 10, 2017 May 10, 2017 DOI: 10.1021/acs.est.7b01272 Environ. Sci. Technol. XXXX, XXX, XXX−XXX

Article

Environmental Science & Technology

horseradish peroxidase (HRP), Amplex Red reacts with H2O2 to produce resorufin, which is a red-fluorescent compound, with excitation and emission wavelengths of 550 and 590 nm, respectively. 2.2.1. Individual Compounds or Their Mixtures. A mixture of DTT (1.2 mL of 1 mM solution, final concentration in the reaction vial = 100 μM), TPT (50 mM; 2.4 mL), and sample (either individual quinones, Fe, Mn, Cu, or their mixtures; 6.4 mL) was incubated at a temperature of 37 °C and pH of 7.4 (maintained by potassium phosphate buffer; 2 mL) in a continuously shaken conical centrifuge vial (presterilized polypropylene tubes) using a thermomixer (Eppendorf). The total reaction volume was 12 mL. At designated time intervals (0, 30, 60, 90, 120, and 180 min for quinones, Fe, and their mixtures, and 0, 60, 120, 180, 240, and 300 min for Cu), 2 mL of the incubating mixture was withdrawn and mixed with 1 mL of 100 mM DMSO to quench the reaction between •OH and TPT.41 The emission intensity of the fluorescent product 2-OHTA was measured by a Shimadzu spectrofluorophotometer (RF-5301pc). The excitation and emission slit widths were each set at 5 nm. Each test was accompanied by a blank (Milli-Q water; resistivity = 18.2MΩ cm), analyzed in the same way as sample by replacing the sample volume with Milli-Q water in the reaction vial. Tests were conducted to confirm that TPT does not interfere with the oxidation of DTT, either in blanks (Milli-Q water) or samples. An independent (unpaired) sample t test showed no significant difference (p > 0.10) between DTT oxidation rates in the reaction mixtures with and without TPT, for both blanks (0.35 ± 0.05 nmol/min without TPT and 0.37 ± 0.04 nmol/min with TPT) and PQ (1.88 ± 0.11 without TPT and 1.90 ± 0.08 with TPT), each analyzed in triplicate. A similar protocol was also used for H2O2 measurement: DTT (0.5 mL, 100 μM), phosphate buffer (1 mL), and sample (3.5 mL) were added in the reaction vial kept at 37 °C. At designated time intervals, 100 μL of the reaction mixture was withdrawn and mixed with the Amplex Red working solution [50 μL of Amplex Red reagent (2.57 mg/mL) + 100 μL of HRP (10 U/mL) + 4.85 mL of phosphate buffer] and kept in an amber vial. The fluorescence of the reaction product was measured after 1 min using the spectrofluorophotometer (RF-5301pc). 2.2.2. Ambient PM2.5 Samples. Ambient PM2.5 samples were collected using a high-volume sampler (HiVol, Thermo Anderson, nondenuded, nominal flow rate 1.13 m3 min−1, PM2.5 impactor) from an urban site, which is located on the roof (height from ground level ∼30 m) of a parking garage in University of Illinois Urbana− Champaign (UIUC) campus (North Campus Parking). The garage is adjacent to University Avenue, which is a major (four-lane) street in the town. The site is about 1 km from downtown Champaign and is surrounded by dense housing and business development. PM at the site is expected to be significantly impacted from the fresh vehicular emissions, as slow moving traffic is typical during the morning and evening rush hour periods, in addition to the construction activities (university dorms). Prebaked (550 °C) 8 in. × 10 in. quartz filters (Pallflex Tissuquartz, Pall Life Sciences) were used to collect the particles. A total of 10 samples along with field blanks were collected from June 27, 2016 to August 12, 2016 (details of date and time are given in Table S1), each for an exact duration of 24 h. The filters after collection were placed immediately in a freezer (temperature = −20 °C) until analyzed for the ROS generation and DTT oxidation. For the measurement of •OH generation and DTT oxidation from PM samples, 20 punches (each 1 in. diameter) were taken from the filter and extracted in 30 mL of Milli-Q water by sonication for 30 min. These extracts were filtered using PTFE syringe filters (0.45 μm pore size; Fisherbrand). Then, 6.4 mL of the extract was used for measuring • OH and the rest was used to measure DTT oxidation in the same manner as standard compounds. Five additional punches from each filter were again extracted in 7.5 mL of Milli-Q water, and 3.5 mL of the filtered extract was used for the measurement of H2O2 generation following the same protocol as for the standard compounds (i.e., quinones and metals). Each sample was analyzed with a corresponding blank filter extracted along with the PM sample, and all the data was blank-corrected. The H2O2 and •OH generation rates from the blank filter extracts (0.33 ± 0.04 μM H2O2/min and 0.13 ± 0.02 nmol of

with several biological end points [hemeoxygenase-1 (HO-1) expression,35 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction activity,36 fraction of nitric oxide in exhaled breath,10 and increased relative risk for asthma12,37 and congestive heart failure]37 in several studies. There are limitations in DTT assay as well, since it does not capture an important step of ROS cascade, which is the generation of •OH. •OH is the most damaging ROS,38 with an in vivo half-life of approximately 10−9 s.39 In the conventional DTT assay protocol, where we measure only the rate of decay of DTT,19 which at most corresponds to the rate of formation of O2•−, the generation of •OH is not captured. This could be one of the caveats causing an underestimation of the role of important metals (e.g., Fe) in DTT assay, which are otherwise known to contribute to ROS generation in biological system.40 In the present study, we overcame this limitation by measuring, for the first time, the rate of •OH generated by ambient particles in DTT assay. To further understand the mechanisms of ROS generation, we tested several quinones (9,10-phenanthrenequinone (PQ), 1,4-naphthoquinone (1,4NQ), 1,2-naphthoquinone (1,2-NQ), and 5-hydroxy-1,4naphthoquinone (5H-1,4NQ)) and metals [Cu(II), Mn(II), Fe(II), and Fe(III)] known to be present in ambient PM for their intrinsic capability to generate H2O2 and •OH in DTT assay. The interaction among certain PM components such as Fe and quinones was also investigated. As a contrast between ROS generation versus DTT consumption, the rate of DTT oxidation was measured from both individual compounds (i.e., quinones and metals) and the ambient PM. The primary objective of this pilot study is to improve the capability of the DTT assay to measure the ROS generation potential of ambient PM by incorporating the contribution from more PM components in a single measurement.

2. EXPERIMENTAL METHODS 2.1. Reagents. Dithiothreitol (assay: ≥98%TLC), 5,5′-dithiobis(2nitrobenzoic acid) (assay: ≥98%), 9,10-phenanthrenequinone (assay: ≥99%), 5-hydroxy-1,4-naphthoquinone (assay: 97%), 1,2-naphthoquinone (assay: 97%), 1,4-naphthoquinone (assay: 97%), potassium phosphate dibasic (assay: ≥98%), potassium phosphate monobasic (assay: ≥98%), iron(II) sulfate heptahydrate (assay: ≥99%), iron(III) chloride (assay: >97%), copper(II) sulfate pentahydrate (assay: ≥98%), and manganese(II) chloride tetrahydrate (assay: ≥98%) were obtained from Sigma-Aldrich Co. (St.Louis, MO). Disodium terephthalate (assay: 99+%) was bought from Alfa Aesar Co. (Haverhill, MA). Amplex Red kit was obtained from Invitrogen Co. (by Thermo Fisher Scientific). All the chemicals bought were of the highest purity available. 2.2. Measurement of •OH and H2O2. A fluorescence-based approach was used to measure both •OH and H2O2 generated in the DTT assay. Son et al.41 reviewed various probes [disodium terephthalate (TPT), 3′-p-(aminophenyl) fluorescein, coumarin-3carboxylic acid, and sodium benzoate] for measuring the •OH catalyzed by ambient PM in the ascorbate assay and recommended TPT as the most appropriate probe based on several criteria (i.e., detection limit, product stability, optimal pH range (6−11), specificity, and yield). We adopted the same approach here and added TPT in the reaction vial for capturing the •OH generated in the DTT assay, which forms 2-hydroxyterephtalic acid (2-OHTA). 2-OHTA is a very stable compound (