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Generation of Reactive Oxygen Species Mediated by Humic-like Substances in Atmospheric Aerosols Peng Lin† and Jian Zhen Yu†,‡,* † ‡
Division of Environment, The Hong Kong University of Science & Technology, Clear Water Bay, Kowloon, Hong Kong, China Department of Chemistry, The Hong Kong University of Science & Technology, Clear Water Bay, Kowloon, Hong Kong, China ABSTRACT: Particulate matter (PM)-mediated reactive oxygen species (ROS) generation has been implicated in health effects posed by PM. Humic-like substances (HULIS) are an unresolved mixture of water-extracted organic compounds from atmospheric aerosol particles or isolated from fog/cloudwater samples. In this study, we use a cell-free dithiothreitol (DTT) assay to measure ROS production mediated by HULIS. The HULIS samples are isolated from aerosols collected at a rural location and a suburban location in the Pearl River Delta, China. In our experiments, ROS activities by residue metal ions in the HULIS fraction are suppressed by including a strong chelating agent in the DTT assay. Under conditions of DTT consumption not exceeding 90%, the HULIS-catalyzed oxidation of DTT follows the zero-order kinetics with respect to DTT concentration, and the rate of DTT oxidation is proportional to the dose of HULIS. The ROS activity of the aerosol HULIS, on a per unit mass basis is 2% of the ROS activity by a reference quinone compound, 1,4-naphthoquinone and exceeds that of two aquatic fulvic acids. The HULIS fraction in the ambient samples tested exhibits comparable ROS activities to the organic solvent extractable fraction, which would contain compounds such as quinones, a known organic compound class capable of catalyzing generation of ROS in cells. HULIS was found to be the major redox active constituent of the water-extractable organic fraction in PM. It is plausible that HULIS contains reversible redox sites, thereby serving as electron carriers to catalyze the formation of ROS. Our work suggests that HULIS could be an active PM component in generating ROS and further work is warranted to characterize its redox properties.
’ INTRODUCTION Ambient particulate matter (PM) has been demonstrated in epidemiological studies to cause adverse health outcomes.13 Many of the adverse health effects of PM are associated with oxidative stress derived from PM-mediated generation of reactive oxygen species (ROS) inside affected cells.46 ROS are oxygencontaining species with strong oxidizing abilities, mainly including superoxide anion (O2•), hydroxyl radical (•OH), hydrogen peroxide (H2O2) and other peroxides.7 A number of studies show that ambient PM 810 and diesel exhaust PM11 can catalyze the generation of ROS. However, knowledge on the range of chemical species in PM responsible for ROS generation is very inadequate, due to our limited knowledge of the chemical composition of PM, especially the myriad of organic compounds that constitute the organic fraction of the PM.12 Among numerous PM constituents, metals7,13,14 and quinoid compounds15 have been identified to be capable of catalyzing the generation of ROS in cells. A more recent study indicated that most of the ROS activities in the organic extract of diesel exhaust particles were associated with quinone-like substances.11 The ionizable metals mediate the formation of ROS through catalyzing Fenton type reactions,6 whereas quinoid compounds can serve as catalysts to transport electrons from biological reducing equivalents like NADPH or ascorbate in cells to dissolved O2 and lead to continuous production of O2• and H2O2.9 r 2011 American Chemical Society
Humic-like substances (HULIS) have recently been recognized to be an abundant class of water-soluble organic compounds in atmospheric PM.16 Our measurements in a rural location in South China showed that HULIS accounted for 60% of water-soluble organic carbon (WSOC).17 HULIS in atmospheric PM possess considerable similarities in structural properties to terrestrial and aquatic humic substances (HS), although they differ in relative carbon distribution among the common functional groups (e.g., carboxyl, carbonyl, hydroxyl, aromatic, aliphatic, etc).18,19 The aquatic HS are known to have biological reactivity in water-dwelling organisms.20,21 Some studies have demonstrated that dissolved HS causes oxidative stress on freshwater organisms.2225 However, HULIS is little studied by the air pollution health research community.26,27 In this study, dithiothreitol (DTT) assay was applied to assess the ROS production mediated by HULIS isolated from rural and suburban ambient PM in the Pearl River Delta (PRD) region, South China. The DTT assay, a chemical based redox-active assay developed by Kumagai et al,28 quantifies catalytically active redox species. DTT plays the role that NADPH or ascorbate plays in cells. Figure 1 illustrates a perceived conceptual diagram Received: October 19, 2010 Accepted: November 1, 2011 Revised: November 1, 2011 Published: November 01, 2011 10362
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Figure 1. Conceptual diagram of HULIS-mediated ROS production and concomitant consumption of DTT.
of an organic compound-mediated ROS production and concomitant consumption of DTT. In the presence of excess DTT, the rate of DTT consumption is proportional to the concentration of the catalytically active redox-active species in the sample (e.g., quinones).28 Li et al.29 demonstrated that the DTT-based chemical reactivity is a quantitative measure of in vitro ROS formation catalyzed by PM samples. They applied this DTT assay to diesel exhaust and ambient PM samples and observed that the DTT assay response was correlated with cellular heme oxygenase-1 (HO-1) expression, a sensitive marker for oxidative stress. A number of other researchers also used the DTT assay to quantify PM-mediated ROS formation.11,30,31
’ EXPERIMENTAL SECTION Aerosol Sampling. Ten ambient samples of PM2.5 (PM of less than 2.5 μm in aerodynamic diameter) were used in this work. Five PM2.5 samples were collected at Yangchun (YC) a rural location and another five were collected at Nansha (NS), a suburban district in Guangzhou in the PRD region, China. The samples were collected onto prebaked quartz filters using a highvolume aerosol sampler (TE-6070 V-BL, Tisch Environmental Inc.). The sampling duration for individual samples ranged from 20 to 36 h. All the samples (except NS070916) were collected in the period between November and January, a time coinciding with local crop harvest season, during which crop residue burning was visually observed. Our chemical analysis also confirmed the influence of biomass burning. The concentration of K+ and K+/ EC (elemental carbon) ratio were 56 times higher than those in the nonharvest season.32 More details about the sampling work can be found in our previous papers.17,32 Apart from the total mass of the HULIS fraction, we do not have knowledge of functional group composition or elemental composition, let alone molecular-level compositions about these HULIS samples. The lack of information on the chemical composition makes it difficult to take one step further so as to linking the ROS activities with certain properties of the HULIS fraction. This in turn diminishes the value of carrying out the ROS activity analysis on a larger sample set. This study is intended to assess the ROS generation potential of atmospheric HULIS and to estimate the order of magnitude of such potential in reference to known redox active components. For this reason, we limited our experiments in this study to the 10 ambient samples collected in two different atmospheric environments to obtain statistic meaningful results for this purpose. HULIS Isolation and Determination. HULIS in aerosol filter samples was first isolated from the other constituents in water extracts using solid phase extraction (SPE), followed
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by quantification by an evaporative light scattering detector (ELSD). The isolation procedure separates water-soluble matter into a hydrophilic fraction (the SPE cartridge effluent) and a hydrophobic fraction (i.e., the HULIS fraction or the eluate fraction). Details on the characteristics and performance of this method have been reported in our previous work.17 In brief, portions (24 cm2) of the high-volume filters were extracted with water in an ultrasonic bath. The ratio of filter sample area to water volume was about 1 cm2 mL1. An aliquot of 10 mL of the extract was acidified to pH 2 using HCl before it was loaded on a SPE cartridge (Oasis HLB, 30 μm, 60 mg/cartridge, Waters). By this SPE separation procedure, compounds bearing aromatic rings and multiple polar functional groups that are protonated at pH 2 are retained on the SPE sorbent. The strongly hydrophilic substances such as major inorganic ions (e.g., K+, Na+, NH4+, Mg2+, Ca2+, Cl, NO3, and SO42‑), low molecular weight organic acids and sugar compounds (e.g., levoglucosan, xylose, and sucrose) pass through the SPE cartridge and appear in the effluent fraction.17 The loaded cartridge was subsequently rinsed with two 1 mL portions of water before elution with 12 mL methanol. The effluent and the eluate fractions of this SPE procedure were collected separately. The eluate was evaporated to dryness at 40C under a gentle stream of N2 and redissolved in 5.0 mL of water and then injected into an ELSD (model 3300, Alltech) for HULIS quantification. In our previous work,17,32 we used methanol containing 2% ammonia as the elution solvent because of its high elution efficiency (i.e., 1.5 mL of methanolammonia solution elutes almost all of the HULIS on the SPE cartridge). However, a recent study showed ammonia may react with carbonyl compounds in laboratory-generated secondary organic aerosols to generate imines.33 This has prompted us to examine possible alteration to HULIS chemical composition by NH3 in the elution solvent. Our experiments showed that DTT reactivity of NAFA solutions were enhanced after the SPE protocols with inclusion of ammonia, indicating probable chemical modification of the HULIS fraction by ammonia. We therefore excluded ammonia from the elution solvent in the SPE step. Instead, the volume of methanol used for elution was increased to 12 mL. The elution efficiency of 12 mL methanol is 84 ( 8% (n = 10) of those achieved by 1.5 mL of ammonia-methanol solution. Our previous study17 showed that ∼40% of the carbon content was collected in the effluent fraction, whereas ∼60% of the carbon content remained with the HULIS fraction. DTT Assay. Dithiothreitol (DTT), diethylene triamine pentaacetic acid (DTPA), 1,4-naphthoquinone (1,4-NQ), and 5,50 dithio-bis(2-nitrobenzoic acid) (DTNB) were purchased from Sigma (St. Louis, MO). Nordic Aquatic Fulvic Acid (NAFA) and Suwannee River Fulvic Acid (SRFA) were obtained from International Humic Substances Society (St. Paul, MN). The procedure of the DTT assay adopted in this study mainly follows that used by Li et al.31 An aliquot of 40 μL of a sample to be assayed is first mixed in a 2 mL vial with 1.0 mL of 0.1 M potassium phosphate buffer (pH 7.40). Some of the DTT assays were designed to inhibit DTT consumption by metal ions. In these assays, DTPA was added to the phosphate buffer to make the final buffer solution contain 1 mM DTPA.11 The 40 μL aliquot samples contained 98214 ng/μL HULIS, or 500 ng/μL aquatic fulvic acid solution, or 2.8 ng/μL 1,4-NQ. A 50 μL aliquot of 0.5 mM DTT solution was then added and the mixture was allowed to react at 37 °C in an oven for 90 min. A 100 μL solution of 1.0 mM DTNB (containing 20 mM DTPA) was added to the reaction solution to generate a colored product before 10363
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Figure 3. Comparison of normalized index of oxidant generation (NIOG) values of 10 ambient samples with those of aquatic fulvic acids. The error bars represent the standard deviations of triplicate measurements of the same samples.
Figure 2. HULIS-catalyzed DTT consumption as a function of (a) HULIS dose in an incubation time of 90 min and (b) incubation time. The error bars represent the standard deviations of triplicate measurements. HULIS used in these experiments were extracted from the aerosol sample NS090112.
absorption (A) measurements at 412 nm were taken using a diode-array spectrophotometer within 30 min. The DTT assay response (RDTT), that is, the percentage of DTT consumed, is computed to be RDTT ¼
A0 A 100 A0
ð1Þ
where Ao is the absorbance due to DTT added in a blank sample of pure water. In the blank sample, DTT is only consumed by dissolved O2.28 The calibration curve confirmed that the absorbance is linearly proportional to the DTT concentrations in the solution (R2 > 0.99) under our experiment conditions. Field blank samples were processed following the same DTT assay procedure. The DTT consumption was only 1.1 ( 0.5% (n = 3) after 90 min incubation. In our subsequent experiments, all the data with DTT consumption less than 1.6% (i.e., average plus one standard deviation of the field blank) were considered to have no DTT activity. Model compounds such as 9, 10-phenanthrenequinone (9,10-PQ) and NAFA were processed following through the same SPE protocol as that for HULIS isolation and subjected to the same DTT assay protocol. Here we chose 9,10PQ instead of 1,4-NQ as a model quinone compound for evaluating the impact of the SPE procedure on ROS activity because the latter is thermally unstable and displayed poor recovery after the drying step under N2 flow. The ROS activities of these two model compounds were found not to be affected by the SPE protocols. Determination of Metal Ions. The concentrations of metal ions in the aerosol water extracts, effluent fractions and eluate fractions (i.e., the HULIS fraction) were measured using an inductively coupled plasma mass spectrometry (ICP-MS, Varian,
Santa Clara, CA). Fourteen metal ions (V, Cr, Mn, Fe, Co, Ni, Cu, Zn, Ag, Al, Hg, Cd, Ba, and Pb) were measured. Their detection limits range from 0.1 (Cd) to 4.3 ng/mL (Fe). These metal ions are either abundant (e.g., Al, Zn, and Pb) in the aerosol samples in this region34 or have been reported to be capable of catalyzing ROS generation in affected cells (e.g., V, Cr, Mn, Fe, Co, Ni, and Cu).7,13,14,35,36
’ RESULTS AND DISCUSSION DTT Consumption Catalyzed by HULIS. Figure 2a shows the HULIS-catalyzed DTT consumption in an incubation time of 90 min as a function of the dose of HULIS used in the assay. The rate of DTT oxidation is linearly proportional to the dose of HULIS added into the incubation solution. In the presence of DTPA, the reaction rate is about 33% lower than that in the absence of DTPA. This difference in the catalytic reaction rate is probably attributed to trace metal ions present in the HULIS fractions (see detailed discussion in next section). DTPA is usually used as a chelating agent of metal ions due to its great ability to form stable coordination complexes with them.37 The time-dependent consumption of DTT catalyzed by HULIS is shown in Figure 2b. This experiment was carried out by adding 5.2 μg HULIS into the buffer solution containing DTT and incubating at 37 °C for times varying from 15 to 700 min. The time-dependent plot is linear when the DTT consumption is less than 90%. In another words, the catalytic reaction rate is constant, that is, the reaction is zero-order with respect to DTT, when the DTT consumption does not exceed 90%. Under these conditions, the catalytic DTT oxidation rate is proportional to the catalyst concentration, that is, the abundance of DTT active moieties in HULIS. To compare the DTT activity of HULIS isolated from different aerosol samples or some other substances, the index of oxidant generation (IOG) is computed using eq 2:
IOG ¼
RDTT tm
ð2Þ
where t is the reaction time (min), m is the target analyte mass (e.g., HULIS, 1,4-NQ, etc) (μg). IOG values are then normalized against the IOG value of 1,4-NQ to derive at normalized IOG (NIOG). 10364
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Figure 4. (a) Metal concentrations in aerosol water extracts and in the effluent fractions. (b) Metal concentrations retained with the HULIS fraction. The error bars indicate the standard variations of five aerosol samples taken at the suburban site.
Figure 3 shows the NIOG values of the ten ambient HULIS samples, ranging from 0.011 to 0.025. By definition, the NIOG of 1,4-NQ is 1. It is clear that HULIS are redox-active and capable of catalyzing ROS generation. The rural and the suburban samples had similar average NIOG values (0.016 versus 0.019). The average NIOG of the combined rural and suburban HULIS samples is 0.018 ( 0.004. That is, the capability in catalyzing ROS generation of the ambient HULIS in the PRD rural and suburban areas is around 2% of that by the reference quinone compound (1,4-NQ), on a per unit mass basis. Two aquatic fulvic acid samples (SRFA and NAFA) were also tested using this DTT assay. Their NIOG values were 0.004 for SRFA and 0.011 for NAFA. This result suggests that the oxidative stress effect of HULIS as measured by the DTT assay exceeds those of aquatic HS. Li et al31 measured the ROS activity by the DTT assay in a fresh diesel emission sample and an aged diesel particle sample and reported their respective NIOG values to be 0.00112 and 0.00131, ∼10 times lower than those for HULIS measured in this work. The DTT assay was also applied to eleven individual watersoluble organic compounds, including phthalic acid, 4-hydroxybenzoic acid, syringic acid, acetosyringone, vanillin, iso-eugenol,
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levoglucosan, sucrose, succinic acid, malic acid, and oxalic acid. These compounds are either known to be directly emitted from fresh biomass burning or are known secondary aerosol constituents. They are water-extractable and some of them partially appear in the HULIS fraction.17 In 90 min after adding these species into the incubation, only a very minor fraction (
