Environ. Sci. Technol. 2009, 43, 4757–4762
PM2.5 Constituents and Oxidative DNA Damage in Humans Y O N G J I E W E I , † I N - K Y U H A N , ‡,§ MIN SHAO,† MIN HU,† J U N F E N G ( J I M ) Z H A N G , * ,‡ A N D X I A O Y A N T A N G * ,† State Key Joint Laboratory of Environmental Simulation and Pollution Control, College of Environmental Sciences and Engineering, Peking University, Beijing 100871, China, Department of Environmental and Occupational Health, University of Medicine and Dentistry of New Jersey (UMDNJ)sSchool of Public Health, 683 Hoes Lane West, Piscataway, NJ 08854, and Johns Hopkins Bloomberg School of Public Health, Department of Environmental Health Sciences, 615 N. Wolfe St., Baltimore, MD 21205
Received November 25, 2008. Revised manuscript received March 10, 2009. Accepted March 13, 2009.
Previous studies suggested that certain constituents of ambient PM2.5 can induce or increase oxidative stress in biological systems. The present study is designed to examine whether exposure to traffic generated particles increases the burden of oxidative stress in humans and to identify specific PM2.5 constituents responsible for pollution-induced oxidative stress. We recruited two nonsmoking security guards who worked at a university campus gate by a heavily trafficked road. Pre- and post-workshift spot urines were collected on each of the 29 days of measurement. Concentrations of PM2.5 mass and 126 chemical species were measured at the worksite and a campus background site simultaneously. Urine samples were analyzed for 8-hydroxy-2′-deoxyguanosine (8-OHdG). Factor analysis and linear mixed-effects regression models were used in statistical analyses. Three clusters of PM2.5 species were identified, including PAHs, metals, and polar organic compounds. Urinary concentrations of 8-OHdG increased by >3 times following an eight-hour workshift in participants. Preworkshift urinary concentrations of 8-OHdG were associated with PM2.5 concentrations at the background site. Post-workshift 8-OHdG concentrations were significantly and positively associated with PM2.5 mass, PAHs, and metals, but not polar organic species, measured at the worksite. Our findings provide direct evidence in humans that PM compositions are important in increasing oxidative stress burdens. Our results support that PAHs and metals are biologically active constituents of PM2.5 with regards to the induction of oxidative DNA damages in the human body.
Introduction Increased concentrations in ambient particulate matter (PM), especially PM with an aerodynamic diameter e2.5 µm (PM2.5), * Corresponding author phone: 8610-6275-1925 (X.T.), 1-732-2355405 (J.Z.); fax: 8610-6275-1927 (X.T.), 1-732-235-4004 (J.Z.); e-mail address:
[email protected] (X.T.),
[email protected] (J.Z.). † Peking University. ‡ University of Medicine and Dentistry of New Jersey (UMDNJ)s School of Public Health. § Johns Hopkins Bloomberg School of Public Health. 10.1021/es803337c CCC: $40.75
Published on Web 04/01/2009
2009 American Chemical Society
are associated with excess morbidity and mortality (1, 2). One of the hypothesized biological mechanisms underlying PM effects observed in epidemiological studies is pulmonary or systemic inflammation that is induced or exacerbated through oxidative stress (OS). It is further hypothesized that PM interacts with biological systems through direct generation of reactive oxygen species from the surface of particles, soluble compounds, such as transition metals and organic compounds, or other agitated processes in bodies (3-5). 8-Hydroxy-2′-deoxyguanosine (8-OHdG), one of the products of OS process, is formed from a hydroxyl radical attack at the C-8 position of deoxyguanosine in DNA (6). 8-OHdG is not metabolized and degraded further after being produced in human body (7). Because 8-OHdG in urine is stable and the measurement is relatively easy, it has been commonly used as a biomarker of oxidative stress (8, 9). Several in vitro studies revealed that the formation of 8-OHdG is induced by transition metals, polycyclic aromatic hydrocarbons (PAHs), and quinone-structure species in both coarse and fine PM (10, 11). In in vivo studies, urinary 8-OHdG concentrations have been associated with concentrations of certain transition metals in fine residual oil fly ash, metal fume, electroplating particles (12, 13), some PAHs emitted from coke ovens (14, 15), asbestos, rubber, and azo-dye (16). Several studies have investigated whether urban ambient PM exposure increases the level of 8-OHdG in urines (17, 18). To our knowledge, however, no studies have examined whether urban ambient PM exposure, characterized by chemical species, increases urinary concentrations of 8-OHdG in humans. Ambient PM is composed of element carbon, metals, inorganic chemicals and organic hydrocarbons originated from various sources. The complexity of the physiochemical properties, chemical compositions or their synergism interactions of PM makes it difficult to attribute OS to any single factor. Previous studies have reported associations between 8-OHdG in urine or lymph and some well-known chemicals contained in particles (e.g., elemental carbon, lead, and PAHs). However, little is known concerning the relationship between urinary 8-OHdG and PM constituents when present as a mixture (e.g., ambient PM) in the atmosphere. In the present study, we attempt to examine the impact of urban PM2.5 constituents on urinary concentrations of 8-OHdG in humans. Considering the recent literature that shows particular importance of urban traffic generated particulate air pollution in relation to adverse cardiopulmonary health effects (19, 20), we used traffic-dominated PM2.5 exposure in the present study.
Experimental Section Study Sites and Subjects. The west gate of Peking University was selected as the study site where air pollution was dominated by vehicular emissions. The gate was located at the curbside of a local road on which approximately 8,000 to 10,000 vehicles traveled per day. The gate was staffed with security guards 24 h per day and 7 days per week on a basis of three 8-h work-shifts. We recruited two security guards, as study subjects, who were nonsmokers and worked together between 12:00 h and 20:00 h everyday during the study period. Subject A was 18 years old and had a body mass index of 19.37 kg/m2. Subject B was 20 years old and had a body mass index of 27.76 kg/m2. The time activity patterns were very similar because they had been working at the same site simultaneously, resting in the same dormitory, eating meals provided by the same refectory of the university. During the study period, the participants were asked not to eat fried and roasted meats and to avoid alcoholic drinks. They were free VOL. 43, NO. 13, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 1. Sampling time scheme. from common cold or flu and did not take any medicines. Informed consent was obtained from each participant. PM2.5 Collection. PM2.5 samples were collected on preweighed Teflon filters and quartz filters for 24 h on each day of measurements during the period of 17 November 2006 to 13 January 2007. Samples were collected simultaneously at two sites. One site was the roof of a six-story building in the middle of the Peking University campus; this site was regarded as the campus background site (Site B). The other site was at the west gate of the campus where the two participants were working as security guards (standing on site all the time) (Site W). We used a high volume sampler (Thermo Anderson, Smyrna, USA; 1.13 m3 · min-1; 230 × 180 mm quartz filter) equipped with a size-selective cyclone (16.70 L · min-1; 47 mm Teflon filter in diameter) at Site B and a four-channel sampler (TH-16A, Wuhan, China; 16.70 L · min-1; 3 channels for 47 mm quartz filter in diameter, 1 channel for 47 mm Teflon filter in diameter) at Site W. Chemical Analysis of PM2.5. More than 100 organic species were extracted and quantified using methods established in previous studies (21, 22). Briefly, a mixture of isotope labeled compounds was used as internal standards. The isotopes were spiked on each filter sample prior to extraction. Sequentially, the spiked filters were placed into a vial with 30 mL of dichloromethane/methanol mixture (CH3Cl2/ CH3OH, 3:1 v/v) and then sonicated for 15 min three times in an ice-bath under room temperature. The solutions were concentrated by rotary evaporator and then further concentrated into 1 mL by a gentle blow of ultrapure nitrogen gas. All the final aliquots were analyzed with a GC (HP6890)-MSD (HP5973) system (Agilent, USA). The GC system was equipped with a 30 m length × 0.25 mm i.d. × 0.25 µm film thickness DB-5 MS capillary column. The temperature program procedures were as follows: oven temperature hold at 60 °C for 10 min, temperature ramp of 10 °C min-1 to 300 °C, then isothermal hold at 300 °C for 26 min. The samples were injected splitless with the injector temperature at 310 °C. The detection limits were between 0.1-4 ng µL-1 and the recoveries were ranged between 80% and 110% (23). The elements were extracted by a microwave digestion process similar to previous studies (24-26). Briefly, Teflon filters, after being cut away the plastic ring supports, were placed into clean Teflon vessels, with a mixture of 3 mL nitric acid (65%), 1 mL hydrochloric acid (38%), and 0.2 mL hydrofluoric acid. The vessels were placed in a microwave (MARS5, CEM Corp., USA) with power of 600 W for digestion. The temperature program for digestion was as follows: ramp to 120 °C in 5 min, ramp to 175 °C in 10 min, hold at 175 °C for 20 min. All samples were analyzed with an ICP-MS system (Agilent 7500c, Agilent, USA). The analytical detection limits were 0.001-0.647 ppb and the recoveries were between 84% and 118.4%. 4758
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Exposure Metrics. We constructed three exposure metrics for the study participates, based on 24-h integrated pollution measurements made at Site W (work site) and Site B (campus background). The participants only spent 8 h per day at the work site and the rest of time elsewhere, we assumed that concentrations measured at Site B better represent exposure concentrations that the participant would have encountered during nonworking hours. Therefore personal time-weighted average exposure may be calculated from the following: ExpA ) ExpB + (ExpW - ExpB)/3 where ExpA is the average accumulated personal exposure, ExpB is 24-h averaged concentration measured at site B and ExpW is 24-h averaged concentration measured at Site W. Each of ExpA, ExpB, and ExpW may serve as a surrogate of participants’ exposure. Urine Collection. A preshift spot urine sample and a postshift spot urine sample were collected every other day on each study participant. Samples were collected on 29 days. The postshift urine samples were collected within half an hour after the end of a work-shift (8:00 p.m.); while the preshift urine samples were collected within an hour before the start of next workshift (12:00 p.m.) (see Figure 1). Specimens were stored at -18 °C until analysis. Analysis of Urine samples for 8-OHdG. Urine samples were analyzed for 8-OHdG using an enzyme-linked immunosorbnent assay (ELISA). The assay kit contained 96-well microplates produced by JICA (Japan Institute for The Control of Aging, Fukuroi, Shizuoka, Japan). The procedure was conducted following the manufacturer’s instruction. Every microplate had its own standard working curve for calculating 8-OHdG concentrations in urines. The detection limit was 0.5 ng/mL. Duplicate ELISA analyses of same samples were performed for every sample, and the variation between the duplicates was less than 20%. Although this ELISA-based method for analyzing urinary 8-OHdG has been commonly used and has undoubtedly several advantages (e.g., high sensitivity, ease of use), there is still a discussion regarding the origin of 8-OHdG and the biological meaning of its presence in urine. In addition, the interpretation of data obtained from ELISA may be difficult because of the crossreactivity of the primary antibody with chemicals structurally similar to 8-OHdG (27, 28). Nevertheless, the present study aims to examine relative changes in 8-OHdG concentrations in response to changes in concentrations of PM constituents and thus should not be affected by potential systematic errors of the method. Urine samples were also analyzed for creatinine; and 8-OHdG measurements were reported as creatinine-adjusted concentrations. Statistical analysis. PM2.5 species were used as explanatory variables in all of our analyses. We used a factor analysis with a varimax rotation method to examine the inter-
TABLE 1. Factors And Grouped Chemicals anthraquinone, pyrene, retene, acenaphthylene, phenanthrene, 2-methylphenanthrene, 9-methylanthracene, fluoranthene, acephenanthrylene, 1-methyl pyrene, chrysene, methyl-fluoranthene, benzo[ghi]flouranthene, cyclopenta[cd]pyrene, benzo[a]anthracene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[j]fluoranthene, perylene, benzo[e]pyrene, benzo[a]pyrene, benzo[ghi]perylene, dibenzo[a,h]anthracene, dibenzo[ae]pyrene, coronene, indeno[1,2,3-cd]pyrene natrium, magnesium, aluminum, potassium, calcium, titanium, vanadium, chromium, manganese, iron, cobalt, barium, thorium, uranium dodecanoic acid, tridecanoic acid, tetradecanoic acid, pentadecanoic acid, hexadecanoic acid, heptadecanoic acid, octadecanoic acid, nonadecanoic acid, eicosanoic acid, hencosanoic acid, docosanoic acid, tricosanoic acid, tetracosanoic acid, pentacosanoic acid, hexacosanoic acid, heptacosanoic aicd, octacosanoic acid, tetradecanol, hexadecanol, octadecanol, eicosanol, levoglucsan, monopalmitin, campesterol
factor 1 (26 species)
factor 2 (14 species)
factor 3 (24 species)
TABLE 2. Concentrations Of PM2.5 Species Clusters By Exposure Metrics exposure metrics
ExpW
ExpB
ExpA
pollutant (µg m-3)
no.
median
25th
75th
IQR
mass of PM2.5 metals PAHs polar organic compounds mass of PM2.5 metals PAHs polar organic compounds mass of PM2.5 metals PAHs polar organic compounds
29 29 29 29 29 29 29 29 29 29 29 29
242.59 18.27 0.76 2.00 104.12 4.86 0.31 0.74 154.87 10.80 0.53 1.19
198.53 15.73 0.55 1.53 72.22 3.91 0.25 0.47 108.18 8.13 0.37 0.90
459.57 28.32 2.35 2.84 204.40 9.01 0.70 0.87 274.47 15.60 1.27 1.51
261.04 12.59 1.80 1.31 132.18 5.10 0.45 0.40 166.29 7.47 0.90 0.61
relationships of explanatory variables and to reduce the number of explanatory variables for further analyses. Factorbased clusters of PM2.5 species were then used in regression analysis to examine the relationships between 8-OHdG and each of the PM2.5 species clusters. Because the study used a repeated measurement design, linear mixed models were used in our regression analysis. We used an analysis of variance model with autoregressive terms. Through the regression, we estimated concentration changes and 95% confidence interval (CI) in 8-OHdG associated with an interquartile range (IQR) increase in concentrations of a PM2.5 species cluster. The level of significance for all analyses was set at 0.05. All statistical analyses were performed using SAS 9.1.3 for windows (SAS Institute Inc., Cary, NC, USA.).
Results Concentrations of PM2.5 Species Clusters. Our chemical speciation analyses targeted ∼200 organic species and 20 elements. Among these species, 126 (107 organic compounds, elemental carbon, and 18 inorganic elements) were consistently identified and quantified in most of the 58 PM2.5 samples collected at both sites B and W. In the factor analysis, 3 factors were determined with the consideration of eigenvalue (>1) and the proportion of common variance (>0.80). The three extracted factors were grouped as PAHs (factor 1), metals (factor 2), and polar organic compounds (factor 3), as shown in Table 1. Factor 1 is composed of 26 PAHs; factor 2 is composed of 5 conventional metals and 9 transition metals. Factor 3 is composed of 17 n-alknoic acids, 4 n-alcohols, and 3 other polar organic compounds. We then calculated the sum of all
TABLE 3. 8-OHdG Concentrations In The Two Subjects (µmol mol-1 Creatinine) post-workshift subject A and B A B
no. mean 58 29 29
6.92 7.22 6.62
std 3.67 4.42 2.78
pre-workshift mean 1.83 1.94 1.71
std 0.52 0.50 0.53
p-value
