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Synergistic and Antagonistic Interactions among the Particulate Matter Components in generating Reactive Oxygen Species based on the Dithiothreitol Assay Haoran Yu, Jinlai Wei, Yilan Cheng, Kiran Subedi, and Vishal Verma Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b04261 • Publication Date (Web): 19 Jan 2018 Downloaded from http://pubs.acs.org on January 20, 2018
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Synergistic and Antagonistic Interactions among the Particulate
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Matter Components in generating Reactive Oxygen Species based on
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the Dithiothreitol Assay
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Haoran Yu1, Jinlai Wei1, Yilan Cheng1, Kiran Subedi2, Vishal Verma1*
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1.
2.
Department of Civil and Environmental Engineering, University of Illinois at Urbana-Champaign, 205 North Mathews Avenue, Urbana, IL, 61801, United States Department of Chemistry, University of Illinois at Urbana-Champaign, 505 S Mathews Avenue, Urbana, IL 61801,
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*Corresponding Author Vishal Verma Assistant Professor, Department of Civil and Environmental Engineering, University of Illinois at Urbana-Champaign 205 N. Mathews Ave Urbana, IL 61801 email:
[email protected] phone: (217) 265-6703
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Abstract
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We assessed the interactions among the particulate matter (PM) components in
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generating the reactive oxygen species (ROS) based on a dithiothreitol (DTT) assay.
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We started with the standard solutions of known redox-active substances, i.e.
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quinones (9,10-phenanthraquinone, 1,2-naphthoquinone, 1,4-naphthoquinone and
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5-hydroxy-1,4-naphthoquinone) and metals [Cu (II), Mn (II) and Fe (II)]. Both DTT
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consumption and hydroxyl radical (•OH) generation were measured in the DTT assay.
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The interactions of Fe were additive with quinones in DTT consumption, but strongly
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synergistic in •OH generation. Cu showed antagonistic interactions with quinones in
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both DTT consumption and •OH generation. Mn interacted synergistically with
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quinones in DTT oxidation, but antagonistically in •OH generation. The nature of the
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interactions of these metals (Fe, Mn and Cu) with ambient humic-like substances
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(HULIS) resembled to that with quinones, although the intensity of interactions were
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weaker in DTT consumption than •OH generation. Finally, we demonstrated that the
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DTT consumption capability of ambient PM can be well explained by HULIS, three
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transition metals (Fe, Mn and Cu), and their interactions. But, •OH generation
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involves a contribution (~50 %) from additional compounds (aliphatic species or
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metals other than Fe, Cu and Mn) present in the hydrophilic PM fraction. The study
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highlights the need to account for the interactions between organic compounds and
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metals, while apportioning the relative contributions of chemical components in the
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PM oxidative potential.
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Key Words: Quinones, Metals, Humic-like substances (HULIS), HULIS-metals
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interaction, Reactive Oxygen Species (ROS), Dithiothreitol (DTT) assay
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1. Introduction
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Air pollution has been linked with numerous adverse health outcomes1-3 and is
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responsible for premature deaths in many developing countries.4-6 Ambient particulate
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matter (PM) is considered an important component of the air pollution, as it has been
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associated with increased morbidity and mortality rate in both long-term7-9 and
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short-term10, 11 exposure. One of the major mechanisms believed to be responsible for
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the aerosol health effects is the oxidative stress caused by different components in
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PM.12-14 Organic and metallic species present in ambient PM could interact with the
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cellular reductants and antioxidants to generate reactive oxygen species (ROS),
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exerting oxidative stress.15, 16 The oxidative potential of PM has been associated with
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multiple disorders, e.g. atherosclerosis, asthma and cardiovascular diseases in many
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studies.17-20
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Several chemical methods, e.g. ascorbic acid (AA) assay,21 dithiothreitol (DTT)
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assay,22 and measurement of ROS in a surrogate lung fluid (SLF),23 have been
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developed to measure the oxidative potential of ambient PM. Among these, the DTT
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assay is the most widely used method because of its sensitivity to many transition
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metals and aromatic organic compounds.24, 25 In this assay, the ambient PM effectively
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catalyzes the transfer of electrons from DTT to oxygen, generating DTT-disulfide and
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superoxide radical (•O2-).26, 27 The traditional endpoint in this assay, i.e. consumption
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of DTT22, which has been shown to be proportional to the generation of •O2-27 and
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H2O2,28
is
driven
by
several
quinones
[9,10-phenanthraquinone
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(PQ),
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1,4-naphthoquinone (1,4-NQ) and 1,2-naphthoquinone (1,2-NQ)] and two transition
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metals (Cu and Mn).25 However, a recent study from our group has shown that the
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consumption rate of DTT does not represent the redox behavior of all components and
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thus cannot be equated to the overall generation of ROS by ambient PM.28 The newer
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endpoint, i.e. hydroxyl radical (•OH) measurement in DTT assay,28 is more effective
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in capturing the contribution of additional metals (e.g. Fe), which mediate ROS
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generation mostly through Fenton reaction. Therefore, measuring both DTT
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consumption and •OH generation in DTT assay are important, as together they can
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provide useful hints on a wider spectrum of the PM oxidative properties.
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Relative contribution of various chemical components in the overall oxidative
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potential of ambient PM is still a matter of debate. A few studies have attempted
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deducing the activity of the ambient PM from the intrinsic oxidative potential of its
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well-known redox-active components, by simple summation.24, 25, 29, 30 However, this
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approach is flawed, as there could be both synergistic and antagonistic interactions
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among the PM components and antioxidants to alter their redox properties. There
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have been very limited studies investigating these interactions. For example, Charrier
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and Anastasio31 found the synergistic effect in •OH generation for the binary mixtures
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of Cu (II) + Fe (II), PQ + Fe (II) and 1,2-NQ + Fe (II), using an SLF extraction
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method. Our previous study also found a synergistic effect of Fe (II) and quinones in
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generating •OH using a DTT assay.28 Ignoring these interactions could lead to highly
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erroneous estimates of the total oxidative potential of ambient PM and the
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contribution of various PM components in ROS generation.
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Here, we study interactions among the PM components in the oxidative potential
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measured by the DTT assay. Both DTT consumption and •OH generation in the DTT
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assay were measured by the individual compounds and their mixtures. We started with
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the interaction of known redox-active substances, i.e. four quinones [PQ, 1,2-NQ,
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1,4-NQ and 5-hydroxyl-1,4-naphthoquinone (5-H-1,4-NQ)] and three transition
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metals (Fe, Mn and Cu). These components are ubiquitously present in ambient
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particles and have been studied by many research groups for their individual oxidative
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properties.21, 24-26,
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resembles with the chemical composition of the organic compounds present in
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ambient particles32-38. Therefore, we used fulvic acid as a surrogate of the mixture of
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atmospheric organic compounds and mixed it with the individual transition metals (Fe,
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Mn and Cu) to measure the activities of the mixtures. Since ambient humic-like
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substance (HULIS) have been shown to be ROS-active in many assays,39-42
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interactions among the HULIS extracted from real world aerosol samples and
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individual transition metals were also studied. Finally, the HULIS and the mixtures of
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transition metals (both extracted from the ambient PM samples) were tested for their
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interactions, and compared with the mixtures of the standard solutions of pure
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compounds. The study presents a framework to systematically investigate the
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interactions among the PM components to enhance or suppress the ROS generation.
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The interaction factors for the PM components obtained from this study compile
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useful data for estimating the net oxidative potential of ambient PM based on its
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measured chemical composition in future studies.
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Fulvic acid extracted from terrestrial or aquatic environments
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2. Materials and Methods
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2.1 Chemicals
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PQ (99%), 1,2-NQ (97%), 1,4-NQ (97%), 5-H-1,4-NQ (97%), 2-hydroxyterephthalic
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acid (2-OHTA, 97%), DTT (99%), dithiobisnitrobenzoic acid (DTNB, 98%), copper
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(II) sulfate pentahydrate (98%), manganese (II) chloride tetrahydrate (98%), iron (II)
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sulfate heptahydrate (99% ACS), potassium phosphate dibasic (98% ACS) and
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potassium phosphate monobasic (99% ACS) were purchased from Sigma-Aldrich.
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Disodium terephthalate (TPT, 99%) was purchased from Alfa Aesar. C-18 silica gel
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(premium grade) was purchased from Sorbent Technologies. Suwannee river fulvic
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acid (SRFA Standard II, 2S101F) was obtained from International Humic Substances
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Society (IHSS). Chelex 100 resin was bought from Bio-Rad. Single element standards
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of 1000ppm concentration used in inductively coupled plasma mass spectrometry
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(ICP-MS) were bought from High Purity Standards.
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2.2 Stock solution preparation
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The stock solutions of PQ, 1,2-NQ and 1,4-NQ were made in DMSO, stored in a
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freezer at -20 °C, and discarded after a month. No significant decay in the activity of
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these quinones was observed during that period. However, owing to the instability
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5-H-1,4-NQ (change in color within a day), its stock solution (in DMSO) was made
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everyday, prior to the experiments. The stock solutions of metals were made in
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deionized water (DI; Milli-Q; resistivity = 18.2 MΩ/cm), stored at 4 °C in the
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refrigerator, and used for at most 2 weeks. The stock solution of SRFA was also
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prepared in DI everyday, prior to the experiments. Potassium phosphate buffer (K-PB;
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pH = 7.4, 0.5 M) was made in DI followed by treatment with Chelex resin to remove
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trace metals. TPT was dissolved in Chelex-treated K-PB. The final solutions for each
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redox-active compound (quinones, metals and SRFA) were prepared on the same day
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of experiment by serially diluting their stock solutions in DI. All the mixtures
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prepared for studying the interactions among quinones, metals, SRFA and HULIS
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were left for 30 minutes at room temperature to simulate a similar condition for
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interactions of the mixture components as in the PM extracts, before measuring the
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DTT consumption and •OH generation activity. Tests were conducted to confirm that
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DMSO at the final concentration levels in the reaction vial (
