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Hydroxyl-radical-dependent DNA damage by ambient particulate matter from contrasting sampling locations
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Environmental Research 101 (2006) 18–24 www.elsevier.com/locate/envres
Hydroxyl-radical-dependent DNA damage by ambient particulate matter from contrasting sampling locations Tingming Shi1, Rodger Duffin2, Paul J.A. Borm, Hui Li, Christel Weishaupt, Roel P.F. Schins Institut fu¨r Umweltmedizinische Forschung an der Heinrich-Heine University Du¨sseldorf gGmbH, auf’m Hennekamp 50, D-40225 Du¨sseldorf, Germany Received 9 March 2005; received in revised form 1 September 2005; accepted 28 September 2005 Available online 17 November 2005
Abstract Exposure to ambient particulate matter (PM) has been reported to be associated with increased respiratory, cardiovascular, and malignant lung disease. Previously we have shown that PM can induce oxidative DNA damage in A549 human lung epithelial cells. The aims of the present study were to investigate the variability of the DNA-damaging properties of PM sampled at different locations and times and to relate the observed effects to the hydroxyl-radical (dOH)-generating activities of these samples. Weekly samples of coarse (10–2.5 mm) and fine (o2.5 mm) PM from four sites (Nordrheim Westfalen, Germany) were analyzed for hydrogen-peroxide-dependent dOH formation using electron paramagnetic resonance and formation of 8-hydroxydeoxyguanosine (8-OHdG) in calf thymus DNA using an immuno-dot-blot assay. DNA strand breakage by fine PM in A549 human lung epithelial cells was quantified using the alkaline comet assay. Both PM size distribution fractions elicited dOH generation and 8-OHdG formations in calf thymus DNA. Significantly higher dOH generation was observed for PM sampled at urban/industrial locations and for coarse PM. Samples of fine PM also caused DNA strand breakage in A549 cells and this damage could be prevented using the hydroxyl-radical scavengers 5,5-dimethyl-1-pyrrolineN-oxide and dimethyl sulfoxide. The observed DNA strand breakage appeared to correlate with the hydroxyl-radical-generating capacities of the PM samples but with different profiles for rural versus urban/industrial samples. In conclusion, when considered at equal mass, dOH formation of PM shows considerable variability with regard to the sampling location and time and is correlated with its ability to cause DNA damage. r 2005 Elsevier Inc. All rights reserved. Keywords: Ambient particulate matter; Electron paramagnetic resonance; DNA damage
1. Introduction Epidemiological studies have demonstrated that increased exposure to ambient particulate matter (PM) is associated with increased respiratory, cardiovascular, and malignant lung disease, thereby increasing morbidity and mortality (Daniels et al., 2000; Pope et al., 2002). However, how exposure to PM can produce the health effects Corresponding author. Fax: +49 211 3389 331.
E-mail address: [email protected] (R.P.F. Schins). Present address: Hubei Provincial Centre for Disease Control and Prevention, Zhuodaoquan North Road 6, 430079 Wuhan, People’s Republic of China. 2 Present address: ELEGI/Colt Laboratory, Centre for Inflammation Research, The Queen’s Medical Research Institute, University of Edinburgh, Edinburgh, Scotland. 1
0013-9351/$ - see front matter r 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.envres.2005.09.005
observed in epidemiological studies and which components of PM mediate such effects are still not clear. Although epidemiological and toxicological evidence suggests that it is the fine (PM2.5) and even the ultrafine (PM0.1) fractions that are responsible for the observed effects, there is no general agreement (Schins et al., 2004; Zhang et al., 2002). The wide range of endpoints suggest that more than one component may be driving the health effects (Dreher, 2000; Donaldson et al., 2003). Some leading hypotheses involve transition metal content and iron mobilization (Molinelli et al., 2002; Aust et al., 2002), particle size and surface (Donaldson et al., 2003), endotoxin contamination (Schins et al., 2004), or organic compounds such as polycyclic aromatic hydrocarbons (Nel et al., 2001). A remarkable consistency with regard to the adverse health effects of PM has been observed throughout many
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epidemiological studies irrespective of the locations where these studies have been carried out, i.e., with different PM composition. However, recent research has demonstrated that the adverse health outcomes vary from city to city and therefore indicate that specific components or properties of PM may be involved (Katsouyanni et al., 2001). Independent studies involving human volunteer exposures to PM indicate that PM-induced health effects may be due to its chemical composition and more specifically to its transition metal content (Ghio and Devlin, 2001; Schaumann et al., 2004). Transition metals are considered to exert their effects predominantly through the formation of hydroxyl radicals (dOH) via the Fenton reaction (Donaldson et al., 1997) and have been implicated in the inflammatory effects (Schaumann et al., 2004; Carter et al., 1997) and in the DNA damaging properties (Prahalad et al., 2001; Knaapen et al., 2002) of PM. We, along with others, have previously demonstrated that PM induces DNA strand breakage and formation of the hydroxyl-radical-specific lesion 8-hydroxy-20 -deoxyguanosine (8-OHdG) (Marnett, 2000) in lung epithelial cells (Prahalad et al., 2001; Knaapen et al., 2000, 2002; Dellinger et al., 2001; Brits et al., 2004). Furthermore, in a recent biomarker study involving Copenhagen students, personal exposure to PM was found to correlate with their lymphocyte 8-OHdG levels (Sorensen et al., 2003). Electron paramagnetic resonance (EPR), which is a wellestablished method to determine the radical generation properties of various dusts, had been applied by us previously to demonstrate that PM generates dOH in close relation with its ability to induce damage to naked DNA in suspension (Knaapen et al., 2002). More recently, we have shown that the dOH-generating properties of PM and its ability to induce oxidative damage in naked DNA varies considerably with sampling time (Shi et al., 2003a, b), but the implications of this variability with regard to biological effects in human lung cells remain to be elucidated. The aims of the present study were to determine the dOH-generating properties of coarse and fine PM collected over time at several contrasting locations within Germany and to relate these to different capacities to induce damage in naked DNA and in cellular DNA. 2. Materials and methods 2.1. Reagents 5,5-Dimethyl-1-pyrroline-N-oxide (DMPO), Hanks’ balanced salt solution (HBSS), ethidium bromide, Dulbecco’s modified Eagle’s medium (DMEM), phosphate-buffered saline (PBS), diaminobenzidine-tetrahydrochloride, desferoxamine, and dimethyl sulfoxide (DMSO) were obtained from Sigma–Aldrich (Taufkirchen, Germany). Hydrogen peroxide (H2O2) was purchased from Fluka (Seelze, Germany). Calf thymus DNA was obtained from Life Technologies Inc. (Gaithersburg, MD, USA). Vectastain-ABC kit was obtained from Vector Laboratories (Burlingame, CA, USA). For EPR experiments, double-distilled deionized water was used.
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2.2. Collection and sample processing of particulate matter Coarse (2.5–10 mm) and fine (o2.5 mm) fractions of PM10 were sampled in weekly intervals at four locations in Nordrhein Westfalen, Germany in the period of February–May 2000. The locations were Borken (Bo) representing a rural site with approximately 40,000 inhabitants (265 inhabitants/km2) and three urbanized/industrialized sites in Dortmund (Do) with approximately 0.6 million inhabitants (population density 2100 inhabitants/km2) and Duisburg (Du-M and Du-B) with 0.5 million inhabitants (2200 inhabitants/km2). The geographical distances between Duisburg and Borken and between Duisburg and Dortmund are both approximately 50 km. Compared with Borken, both Duisburg and Dortmund are characterized by higher levels of NOx and CO. These locations are included in past and ongoing epidemiological studies on air quality and health (Polat et al., 2002). Coarse and fine PM were collected on preweighed Teflon filters using Graseby–Anderson dichotomous lowvolume samplers at a flow of 16.7 L/min. Filters were stored in the dark in a dry atmosphere until further analysis. The PM was removed from the filters by agitation (5 min) in 1 mL of ultrapure water, followed by sonication for 5 min. Resulting PM concentrations were immediately estimated using comparative turbidometry against a standard dilution curve using a carbon-black suspension. Although this method represents only an indirect estimate of the extracted amount of PM, it allowed for the analysis of freshly prepared (i.e., nonfrozen) PM suspensions. This approach avoids prolonged storage and freeze-thawing of PM suspensions, which can lead to altered leaching of metals or organic constituents and which also could affect particle agglomeration (Donaldson et al., 1997). Upon reconstitution of the filters from which the PM was extracted, the actual extracted mass was also determined gravimetrically, to allow a comparison with the turbidometry determinations. Particle suspensions were diluted in ultrapure water at the concentrations shown and immediately used for analysis of dOH generation, 8-OHdG formation, and DNA strand breakage experiments. Samples were all adjusted to equal concentrations, i.e., to 320 mg/mL for coarse PM and 380 mg/mL for fine PM. For determination of the regional variability of dOH and 8-OHdG formation, pairs of fine and coarse PM were analyzed randomly with regard to sampling period and sampling location. A random selection of fine PM was also used for the determination of DNA strand breaks in A549 human lung epithelial cells.
2.3. Electron paramagnetic resonance Hydroxyl radical formation by the coarse and fine PM was evaluated by EPR as described previously (Shi et al., 2003a, b). Briefly, 50 mL of the freshly prepared particle suspension was mixed with 100 mL of the spin trap DMPO (0.05 M in distilled deionized water) and 50 mL H2O2 (0.5 M in PBS). The suspension was incubated for 10 min at 37 1C in a shaking water bath and filtered through a 0.1 mm filter (Acrodisc 25 mm syringe filter, Pall Gelman Laboratory, Ann Arbor, MT, USA). The filtrate was immediately transferred to a capillary and measured with a Miniscope EPR spectrometer (Magnettech). The EPR spectra were recorded at room temperature using the following instrumental conditions: magnetic field, 3360 G; sweep width, 100 G; scan time, 30 s; number of scans, 3; modulation amplitude, 1.975 G; receiver gain, 1000. Quantification was done by accumulation of three spectra, each averaging three scans. All four peaks were quantified by measuring the amplitudes, and outcomes are expressed as the total amplitude in arbitrary units (AU). Comparative evaluation of this quantification protocol with the method of double integration as usually applied in EPR studies demonstrated nearly identical results for DMPO-OH spectra (n ¼ 30, Pearson’s r ¼ 0:99, Po0:001).
2.4. Hydroxydeoxyguanosine induction by PM in calf thymus DNA Induction of the dOH-specific DNA lesion 8-hydroxydeoxyguanosine by PM in isolated calf thymus DNA was estimated via a dot-blot assay
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that we have developed recently (Knaapen et al., 2002) based on a method described by Musarrat and Wani (1994). Freshly prepared suspensions of PM were incubated with 50 mg of calf thymus DNA dissolved in Tris–HCl (10 mM, pH 8.0) and H2O2 (1 mM). In each experiment, DNA incubated without PM and H2O2, and DNA incubated with 0.1 mM FeSO4 and 1 mM H2O2 were included, respectively, as negative and positive controls. Samples were incubated in the dark for 90 min at 37 1C in a shaking water bath and then immediately centrifuged (6000 rpm, 5 min). Then 200 mL of the supernatant was transferred to a fresh tube, and DNA was precipitated by the addition of 1/10 vol. NaAc (1.5 M, pH 6.0) and 2 vols 100% icecold ethanol at 20 1C for 1 h. The DNA was then washed twice using 70% ethanol (13,000 rpm, 5 min), dried in the dark, dissolved in 30 mL of Tris–HCl buffer, and stored overnight at 4 1C. DNA concentrations were determined spectrophotometrically and the samples were diluted to a final concentration of 2.56 mg/mL in 20 standard saline citrate. Of each sample, replicate two-fold dilutions were blotted on a nitrocellulose membrane using a dot-blot apparatus. To each blot, both negative and positive controls were added. The DNA was cross-linked by baking the membrane for 90 min in a prewarmed oven at 80 1C. Blocking of the membrane was performed overnight using casein. Immuno-localization of 8-OHdG was performed using the N45.1 monoclonal antibody (Toyokuni et al., 1997) and the Vectorstain-ABC kit with diaminobenzidine staining according to the recommended protocol (Vector Laboratories). The blots were analyzed by computer-assisted densitometry scanning (Bio-Rad) and expressed relative to the density of the negative controls. The relative staining intensities as ranked for each separate dot-blot experiment were used for statistical evaluation.
2.5. Treatment of A549 cells with fine PM A549 cells (American Type Culture Collection) were grown in DMEM supplemented with 10% heat-inactivated fetal calf serum, L-glutamine, and 30 IU/mL penicillin–streptomycin at 37 1C and 5% CO2. For experiments, cells were trypsinized at confluence, seeded into 60-mm culture dishes, and grown until confluence. Cells were washed two times with HBSS and then treated with freshly prepared samples of fine PM as described before upon dilution in HBSS at a final concentration of 20 mg/ cm2. Cells were incubated for 3 h at 37 1C (100% relative humidity, 5% CO2). A total of 15 fine PM samples were randomly selected three independent experiments measuring each sample in duplicate. In additional experiments, DMSO and DMPO were used as hydroxyl radical scavengers.
2.6. DNA strand break analysis DNA strand break formation in A549 cells was determined by the alkaline comet assay (Tice et al., 2000), as described previously (Schins et al., 2002). Fully frosted slides were covered with a layer of 0.65% agarose using a cover slide and stored overnight at 4 1C. Following treatment, A549 cells were harvested from the dishes using trypsin and were then suspended in HBSS. Cytotoxicity in A549 cells caused by exposure procedures and cell processing was evaluated using Trypan blue dye exclusion. Subsequently, 25 ml of the cell suspension (approximately 2 106 cells/mL) was mixed with 75 mL 0.5% low-melting-point agarose. This mixture was then added to the slides on top of the first agarose layer using a cover glass. Slides were stored 45 min at 4 1C to allow solidification and covered with another layer of low-melting-point agarose (100 mL). Following solidification for at least 45 min at 4 1C, slides were immersed in lysis buffer (2.5 M NaCl, 100 mM EDTA, 10 mM Tris base, 1% sodium lauryl sarcosinate, pH 10, 10% DMSO, and 1% Triton X-100 added just before use) and stored overnight at 4 1C. The following day, slides were rinsed with distilled water and placed in an electrophoresis tank filled with ice-cold buffer (300 mM NaOH, 1 mM EDTA, pH 13) for 30 min. Electrophoresis was conducted at 300 mA and 25 V for 15 min. Slides were then neutralized 3 10 min using neutralization buffer (0.4 M Tris, pH 7.5). All steps described were performed in the dark or under dimmed red light to prevent additional DNA damage. Slides were stained with
ethidium bromide (20 mg/mL in H2O) and comet appearances were analyzed using an Olympus BX60 fluorescence microscope at 1000 magnification. On every single slide 50 cells were analyzed randomly and classified into one of five categories according to tail length (0, 1, 2, 3, 4, in which 0 ¼ no tail). For final analysis a comet score of each individual slide was calculated according to the method described in Knaapen et al. (2002): comet score ¼ sum (class 1 cells+2x class 2 cells+3x class 3 cells+4x class 4 cells). Using this formula, a minimally damaged sample will have a score of 0, whereas a maximally damaged sample obtains a comet score of 200. In further experiments, comet appearances were also evaluated using the comet image analysis software program Comet Assay II (Perceptive Instruments, Haverhill, UK).
2.7. Statistical analysis Comparisons between fine and the coarse PM or of the PM samples of different sampling sites (rural area (Bo) versus other sites) were carried out using the t-test or the nonparametric Mann–Whitney u test (for 8-OHdG only). All correlation was determined using simple linear regression analysis.
3. Results The mean masses of the PM sampled at the different locations and extracted from the Teflon filters using our protocol are summarized in Table 1. The sampled masses and the extracted masses were remarkably similar for the four locations, with the exception of coarse PM from Dortmund, which was significantly higher than coarse PM from Borken. Using our extraction protocol (vortexing and sonication) the mass recovered from the Teflon filters appeared to be quite high for the coarse fraction (mean: 97%; range: 84–105%) and the fine fraction (mean: 89%; range: 86–92%). For both PM size fractions, the recovery appeared to be a constant proportion of the filter loading as indicated by a linear correlation between the sampled and the recovered mass (fine: r ¼ 0:92, Po0:001; coarse: r ¼ 0:69, Po0:001). Since turbidometry is the only method that can be used to estimate mass for immediate use with small amounts of PM, we analyzed the relationship between gravimetric and turbidometric data and found a highly significant correlation between mass and density for both the fine and coarse fractions. Similar correlation coefficients were found for the fine (r ¼ 0:58, n ¼ 40, Po0:01) and the coarse (r ¼ 0:41, n ¼ 41, Po0:01) fractions, but the slope of the line shows that, in our hands, the concentrations expressed by turbidometry lead to an underestimation (30%) of extracted mass. Since hydroxyl radicals produced via Fenton-like reactions have been considered key features of PM, we used EPR with spin-trapping to quantify dOH formation by PM as an entirety. All PM samples collected during this study showed the typical spectrum for DMPO-OH, differing only in signal amplitude of dOH formation. The quantification of the EPR measurements is shown in Fig. 1. As can be seen in the figure, urban PM had significantly higher dOH generation capacity than rural PM. The coarse fractions generally showed a higher signal than the fine
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Table 1 Characterisation of fine (1a) and coarse (1b) PM sampled at four different locations Borken
Dortmund
Duisburg-meid
Fine PM Number of samples Sampled mass (mg) Extracted mass (mg) Recovery (%) Estimation of extracted PM (mg/mL) (turbidometry)
12 2.270.6 2.070.6 88.9711.6 0.570.1
7 2.770.7 2.570.6 92.374.8 1.070.4
Coarse PM Number of samples Sampled mass (mg) Extracted mass (mg) Recovery (%) Estimation of extracted PM (mg/mL) (turbidometry)
14 1.870.5 1.670.5 93.5714.3 0.570.2
7 2.771.0 b 2.770.9 b 98.377.7 0.670.1
Duisburg-bus
14 2.370.8 2.170.8 89.4724.5 0.670.2
7 2.170.7 1.870.7 86.2710.3 0.670.2
14 2.371.1 2.070.6 105.1746 0.670.3
6 2.371.0 1.870.7 84.1729.3 0.670.2
160
EPR Signal (A.U)
140
fine Coarse
* **
120 100
**
**
80
EPR Signal (A.U)
All data are expressed as mean7standard deviation.
*
60
180 160 140 120 100 80 60 40 20 0 8
40
9
10
11
12
13
14
15
16
17
18
19
20
21
18
19
20
21
Sampling Weeks
(A)
0
Bo
Do
Du-M
Du-B
Fig. 1. Hydroxyl radical generation of coarse and fine PM of suspensions sampled at four locations. Data are expressed mean and SD of the intensity of the resulting DMPO-OH signal in arbitrary units from the weekly samples of coarse and fine PM. PM samples from the urban/ industrial areas (Do, Du-M, Du-B) showed higher ability to generate hydroxyl radical than the PM samples from the rural location (Bo). The asterisks indicate statistically significant differences from the rural location (Bo), at Po0:05 and Po0:01.
fractions, with the exception of samples from Dortmund. In addition to differences in both PM size and sampling location, considerable temporal variability in dOH formation was observed. Fig. 2 shows, as an example, the dOH generation for consecutive sampling weeks of both fine and coarse PM from the rural location (Bo) and an urban location (Du-M). As can be seen in the figure, irrespective of the week-to-week variations of the dOH signals, coarse PM consistently showed higher ability to generate dOH than fine PM for these locations. To evaluate whether the observed variability in dOH generation relates to the induction of 8-OHdG, coarse and fine fractions of PM were incubated with calf thymus DNA in the presence of 1 mM H2O2 and analyzed using an immuno-dot-blot assay. Both fine and coarse PM caused formation of 8-OHdG and the effects tended to be stronger for the PM samples collected at the urban/industrialized locations (Fig. 3). However, significantly higher 8-OHdG
EPR Signal (A.U)
20
(B)
180 160 140 120 100 80 60 40 20 0
8
9
10
11
12
13
14
15
16
17
Sampling Weeks
Fig. 2. Time course of week means of DMPO-OH formation of the fine (A) and the coarse (B) fraction of PM as sampled in the rural town (Bo) (m) and a location of the city of Duisburg (Du-M) (’). Data are expressed as the intensity of the resulting DMPO-OH signal in arbitrary units. PM sampled at the urban area cause higher ability to generate hydroxyl radical. Variations were observed for both fractions and locations, large variations were found among urban samples.
was observed only with coarse PM from Duisburg-M compared to coarse PM from the rural location Borken. A significant correlation was found between the dOH generation as determined by EPR and the induction of 8OHdG (n ¼ 81, Spearman’s r ¼ 0:48, Po0:001). To determine the significance of these observations for cellular DNA damage, A549 cells were treated with fine PM samples, randomly selected from both urban and rural locations. DNA strand breakage results are presented in Fig. 4. As can be seen in Fig. 4A, no significant differences between the different sampling locations were found in DNA damage, but a considerable variability among the
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5
3 2
4 3
1
1
0
0 Do
Du-M Du-B
40 20 0
Bo
Do Du-M Du-B
DNA damage (comet score)
DNA damage (comet score)
(A)
60
(B)
10
#
##
PM & DMPO
PM & DMSO
#
5 0 control
PM
DMPO
DMSO
3 ** Bo
Do
Du-M Du-B
Fig. 3. Induction of 8-OHdG by fine and coarse fractions of PM in calf thymus DNA. The data shown are ranked 8-OHdG in accordance with the relative staining intensities for each separate experiment (ranks 1–6). For each sampling location the horizontal line represents the median value. Urban area PM demonstrated a higher ability to induce 8-OHdG than rural PM. Significant differences were observed only in urban PM (Du-M) (Po0:05) compared to rural particles (Bo).
80
**
2
tail moment
4
tail intensity (%)
5
Bo
15
Coarse PM 6
8-OHdG (rank)
8-OHdG (rank)
Fine PM 6
2 ## 1
##
#
##
0 control
PM
PM & DMPO
PM & DMSO
DMPO
DMSO
Fig. 5. DNA strand breakage by fine PM in the presence or absence of the hydroxyl radical scavengers DMPO and DMSO. A549 cells were treated for 3 h with PM suspended in HBSS (300 mg/mL) and in the presence or absence of DMPO (15 mM) or DMSO (0.5% in v/v). DNA damage as analyzed by a comet image analysis software program are expressed as % tail DNA (A), and tail moment (B) (data are mean and standard deviation; n ¼ 4). Both scavengers caused significant inhibition of PMinduced DNA strand breakage, whereas the scavengers alone had no effect on DNA strand breakage ( Po0:01 versus control; ]Po0:05 versus PM; ]]Po0:01 versus PM).
80 60 40 20 0 20
40
60
80
DMPO-OH (A.U)
Fig. 4. Induction of DNA strand breakage by fine PM from different sampling locations in A549 cells (A) and its relation with hydroxyl generation (B). A549 cells were treated for 3 h with fine PM samples from different locations at equal doses (20 mg/cm2). No significant differences in DNA damage were found in the different sampling locations (A). When samples were subdivided into rural and urban/industrial locations (B), different slopes were observed in the hydroxyl-radical-generating ability of the samples as determined by EPR and the induction of DNA strand breakage in A549 cells, respectively, for rural samples (J) versus urban samples (K).
samples, in line with the dOH-generating properties of the different samples, was observed. In Fig. 4B, the dOHgenerating capacities of the PM samples tended to correlate with their ability to elicit DNA strand breakage in the A549 cells. When samples were subdivided into rural and urban samples, clear associations between dOH generation and DNA strand breakage were observed albeit with different slopes. To evaluate whether dOH generation, as determined by EPR, may indeed be involved in the observed DNAdamaging effects of PM, subsequent experiments using the hydroxyl-radical scavengers DMSO and DMPO were performed. The effects of DMSO and DMPO on DNA strand breakage by fine PM are shown in Fig. 5. Here, coincubation with both DMSO and DMPO caused significant reductions of DNA strand breakage in the A549 cells.
4. Discussion The data in our present study are in line with earlier observations that PM can elicit DNA damage in lung epithelial cells via inherent oxidative properties. It is generally accepted that reactive oxygen species (ROS) are able to cause DNA strand breaks and DNA oxidation, processes that have been implicated in the initiation stage of carcinogenesis (Marnett, 2000). Among the major products of oxidative DNA damage is the premutagenic lesion 8-hydroxy-20 -deoxyguanosine (Marnett, 2000; Collins et al., 1993). Indeed, a clear correlation between hydroxyl-radical generation by PM and induction of 8OHdG in naked DNA was observed in our study, which was also in line with our previous observations (Shi et al., 2003a, b). Obviously and in contrast to acellular methods such as EPR and naked DNA damage assays, the oxidative DNAdamaging properties of PM in cellular systems involves more complex biochemical and physical processes, where various constituents of PM may interact with components on the surface and inside the cell. The EPR method and the 8-OHdG assays employed in this research are both performed in the presence of exogenous hydrogen peroxide and therefore most likely reflect the Fenton reactivity of the samples. Several other mechanisms have been considered, e.g., generation of ROS such as hydroxyl radical by PM as a result from its surface area (Kuchino et al., 1987) and
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organic constituents (Nel et al., 2001; Brown et al., 2001). It has been found that dOH generation may also be modified by other organic and inorganic components, such as sulfates (Li et al., 2002) and semiquinones (Dellinger et al., 2001). Previously, we and others have shown that PM induces both DNA strand breaks and 8-OHdG in lung epithelial cells (Prahalad et al., 2001; Knaapen et al., 2000, 2002; Dellinger et al., 2001; Brits et al., 2004; Ghio et al., 1999). Inhibition of PM-induced DNA strand breakage with hydroxyl-radical scavengers such as tetramethylthiourea or dimethyl sulfoxide, the antioxidant enzyme catalase, and the iron chelator deferoxamine suggest that dOH produced by the Fenton reaction may play a predominant role in these cellular systems (Knaapen et al., 2002; Dellinger et al., 2001). The data from our present study support these observations, since for fine PM a correlation between DNA damage in A549 cells and hydroxyl-radical generation within this PM fraction was observed. Furthermore, it was found that the hydroxylradical scavengers DMPO and DMSO were capable of inhibiting strand breakage by fine PM. With regard to the current effects as observed with fine PM, we have recently demonstrated, using immunocytochemistry, that this fraction and coarse PM elicit 8-OHdG formation in A549 cells (Shi et al., 2003a, b). However, in that study we were not able to correlate dOH generation to this oxidative DNA lesion, due to the semiquantitative nature of the method. Quantitative analysis of 8-OHdG, e.g., using high-performance liquid chromatography with electrochemical detection (Don Porto Carero et al., 2001), necessitates incubations of large cell numbers to obtain sufficient DNA and therefore high mass of PM. In this respect, a more useful approach for the quantitative detection of DNA damage, i.e., the alkaline comet assay which requires relatively low cell numbers for analysis (Tice et al., 2000) was used and this was suitable in our hands given the limited collected amount of PM in our setup. Recently developed modifications of the comet assay, which, for example, allow specific identification of oxidative lesions such as 8-OHdG (Schins et al., 1995), were unfortunately not routine in our lab at the time of measurement. Nevertheless, taken together, our previous studies with 8OHdG (Shi et al., 2003a, b) and our current observations on DNA strand breakage in relation to EPR measurements and the effect of DMSO and DMPO indicate that the comet assay represents an indicator of oxidative stress by PM in A549 cells. Importantly, we showed that PM elicits DNA strand breakage in cultured cells in the absence of exogenous hydrogen peroxide, whereas hydroxyl-radical generation by PM using EPR has been measured upon addition of a rather high concentration of H2O2. Previously, we showed that micromolar levels of H2O2, concentrations occurring in physiological conditions, already enhance hydroxyl radical generation by PM (Knaapen et al., 2002; Shi et al., 2003a, b). Complementary to our EPR work, EPR measurements of dry PM samples also show spectra
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indicative of semiquinone radicals similar to those found in cigarette tar samples, suggesting that H2O2 formation via quinone cycling (Dellinger et al., 2001) can attribute to dOH generation. Our data demonstrate that, although PM samples from different areas have the ability to generate hydroxyl radicals and cause DNA damage, the patterns may differ between sampling locations. On the one hand, PM samples from an industrial/urban location showed higher ability for the formation of hydroxyl radicals than rural PM compared at equal mass. On the other hand, no clear differences were found in the induction of 8-OHdG in naked DNA or DNA strand breakage in lung epithelial cells by PM samples from different locations. Despite these observations, for both rural and urban samples, DNA damage appeared to increase with increasing hydroxylradical-generating capacities. Obviously, however, it should be emphasized that our results are observed with a relatively low number of PM samples and these results should be interpreted with some caution. Therefore further studies are needed to confirm the contribution of hydroxylradical generation toward the induction of DNA damage. In conclusion, we have demonstrated that PM, both coarse and fine, sampled over time and at different locations have the ability to generate hydroxyl radicals, induce 8-OHdG formation in calf thymus DNA, and elicit DNA strand breaks in human lung epithelial cells. Importantly, PM shows considerable variability in each of these endpoints with regard to the sampling location and time. DNA strand breakage appeared to correlate with the hydroxyl-radical-generating capacities of the PM samples but with different profiles for rural versus urban/industrial samples. The observed location-specific characteristics of PM are in agreement with recent observations in epidemiological research (Katsouyanni et al., 2001) and forward a message that geographic or site-related physical–chemical characteristics of PM impact its ability to elicit cellular oxidative stress and DNA damage via mechanisms involving dOH generation.
Acknowledgments The PM samples used for this study have been collected within the framework of the so-called HOTSPOT study, a health surveillance program in Nordrhein Westfalen, supported by the Landesumweltamt (LUA) in Essen, Germany. We acknowledge Ms. Olschewski and Mr. Schwarz from the LUA for their assistance in PM sampling.
References Aust, A.E., Ball, J.C., Hu, A.A., Lighty, J.S., Smith, K.R., Straccia, A.M., Veranth, J.M., Young, W.C., 2002. Particle characteristics responsible for effects on human lung epithelial cells. Res. Rep. Health Eff. Inst. 110, 1–76.
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ambient concentrated and diesel exhaust particulate matter. Inhal. Toxicol. 14 (5), 459–486. Marnett, L.J., 2000. Oxyradicals and DNA damage. Carcinogenesis 21, 316–370. Molinelli, A.R., Madden, M.C., McGee, J.K., Stonehuerner, J.G., Ghio, A.J., 2002. Effect of metal removal on the toxicity of airborne particulate matter from the Utah Valley. Inhal. Toxicol. 14 (10), 1069–1086. Musarrat, J., Wani, A.A., 1994. Quantitative immunoanalysis of promutagenic 8-hydroxy-20 -deoxyguanosine in oxidized DNA. Carcinogenesis 15, 2037–2043. Nel, A.E., Diaz-Sanchez, D., Li, N., 2001. The role of particulate matter in pulmonary inflammation and asthma: evidence for the involvement of organic chemicals and oxidative stress. Curr. Opin. Pulm. Med. 7 (1), 20–26. Polat, D., Eberwein, G., Becker, A., Weishaupt, C., Schins, R.P.F., Ranft, U., Borm, P.J.A., 2002. Ambient exposure and nasal inflammation in adults and children—a preliminary analysis. Int. J. Hyg. Environ. Health 205, 229–234. Pope III, C.A., Burnett, R.T., Thun, M.J., Calle, E.E., Krewski, D., Ito, K., Thurston, G.D., 2002. Lung cancer, cardiopulmonary mortality, and long-term exposure to fine particulate air pollution. J. Am. Med. Assoc. 287, 1132–1141. Prahalad, A.K., Inmon, J., Dailey, L.A., Madden, M.C., Ghio, A.J., Gallagher, J.E., 2001. Air pollution particles mediated oxidative DNA base damage in a cell free system and in human airway epithelial cells in relation to particulate metal content and bioreactivity. Chem. Res. Toxicol. 14 (7), 879–887. Schaumann, F., Borm, P.J., Herbrich, A., Knoch, J., Pitz, M., Schins, R.P., Luettig, B., Hohlfeld, J.M., Heinrich, J., Krug, N., 2004. Metalrich ambient particles (particulate matter 2.5) cause airway inflammation in healthy subjects. Am. J. Respir. Crit. Care Med. 170, 898–903. Schins, R.P., Knaapen, A.M., Cakmak, G.D., Shi, T., Weishaupt, C., Borm, P.J., 2002. Oxidant-induced DNA damage by quartz in alveolar epithelial cells. Mutat. Res. 517, 77–86. Schins, R.P.F., Schilderman, P.A.E.L., Borm, P.J.A., 1995. Oxidative DNA-damage in peripheral blood lymphocytes of coal workers. Int. Arch. Occup. Environ. Health 67, 153–157. Schins, R.P.F., Lightbody, J., Borm, P.J.A., Donaldson, K., Stone, V., 2004. Inflammatory effects of coarse and fine particulate matter in relation to chemical and biological constituents. Toxicol. Appl. Pharmacol. 195, 1–11. Shi, T., Knaapen, A.M., Begerow, J., Birmili, W., Borm, P.J.A., Schins, R.P.F., 2003a. Temporal variation of hydroxyl radical generation and 8-hydroxy-20 -deoxyguanosine formation by coarse and fine particulate matter. Occup. Environ. Med. 69, 322–329. Shi, T., Schins, R.P.F., Knaapen, A.M., Kuhlbusch, T., Pitz, M., Heinrich, J., Borm, P.J.A., 2003b. Hydroxyl radical generation by electron paramagnetic resonance as a new method to monitor ambient particulate matter composition. J. Environ. Monit. 5, 550–556. Sorensen, M., Autrup, H., Hertel, O., Wallin, H., Knudsen, L.E., Loft, S., 2003. Personal exposure to PM2.5 and biomarkers of DNA damage. Cancer Epidemiol. Biomarkers Prev. 12, 191–196. Tice, R.R., Agurell, E., Anderson, D., Burlinson, B., Hartmann, A., Kobayashi, H., Miyamae, Y., Rojas, E., Ryu, J.C., Sasaki, Y.F., 2000. Single cell gel/comet assay: guidelines for in vitro and in vivo genetic toxicology testing. Environ. Mol. Mutagen. 35, 206–221. Toyokuni, S., Tanaka, T., Hattori, Y., Nishiyama, Y., Yoshida, A., Uchida, K., Hiai, H., Ochi, H., Osawa, T., 1997. Quantitative immunohistochemical determination of 8-hydroxy-20 -deoxyguanosine by a monoclonal antibody N45.1: its application to ferric nitrolotriacetate-induced renal carcinogenesis model. Lab. Invest. 76, 365–374. Zhang, J.J., Hu, W., Wie, F., Wu, G., Korn, L.R., Chapman, R.S., 2002. Children’s respiratory morbidity prevalence in relation to air pollution in four Chinese cities. Environ. Health Perspect. 110 (9), 961–967.
Environmental Research 101 (2006) 18–24 www.elsevier.com/locate/envres
Hydroxyl-radical-dependent DNA damage by ambient particulate matter from contrasting sampling locations Tingming Shi1, Rodger Duffin2, Paul J.A. Borm, Hui Li, Christel Weishaupt, Roel P.F. Schins Institut fu¨r Umweltmedizinische Forschung an der Heinrich-Heine University Du¨sseldorf gGmbH, auf’m Hennekamp 50, D-40225 Du¨sseldorf, Germany Received 9 March 2005; received in revised form 1 September 2005; accepted 28 September 2005 Available online 17 November 2005
Abstract Exposure to ambient particulate matter (PM) has been reported to be associated with increased respiratory, cardiovascular, and malignant lung disease. Previously we have shown that PM can induce oxidative DNA damage in A549 human lung epithelial cells. The aims of the present study were to investigate the variability of the DNA-damaging properties of PM sampled at different locations and times and to relate the observed effects to the hydroxyl-radical (dOH)-generating activities of these samples. Weekly samples of coarse (10–2.5 mm) and fine (o2.5 mm) PM from four sites (Nordrheim Westfalen, Germany) were analyzed for hydrogen-peroxide-dependent dOH formation using electron paramagnetic resonance and formation of 8-hydroxydeoxyguanosine (8-OHdG) in calf thymus DNA using an immuno-dot-blot assay. DNA strand breakage by fine PM in A549 human lung epithelial cells was quantified using the alkaline comet assay. Both PM size distribution fractions elicited dOH generation and 8-OHdG formations in calf thymus DNA. Significantly higher dOH generation was observed for PM sampled at urban/industrial locations and for coarse PM. Samples of fine PM also caused DNA strand breakage in A549 cells and this damage could be prevented using the hydroxyl-radical scavengers 5,5-dimethyl-1-pyrrolineN-oxide and dimethyl sulfoxide. The observed DNA strand breakage appeared to correlate with the hydroxyl-radical-generating capacities of the PM samples but with different profiles for rural versus urban/industrial samples. In conclusion, when considered at equal mass, dOH formation of PM shows considerable variability with regard to the sampling location and time and is correlated with its ability to cause DNA damage. r 2005 Elsevier Inc. All rights reserved. Keywords: Ambient particulate matter; Electron paramagnetic resonance; DNA damage
1. Introduction Epidemiological studies have demonstrated that increased exposure to ambient particulate matter (PM) is associated with increased respiratory, cardiovascular, and malignant lung disease, thereby increasing morbidity and mortality (Daniels et al., 2000; Pope et al., 2002). However, how exposure to PM can produce the health effects Corresponding author. Fax: +49 211 3389 331.
E-mail address: [email protected] (R.P.F. Schins). Present address: Hubei Provincial Centre for Disease Control and Prevention, Zhuodaoquan North Road 6, 430079 Wuhan, People’s Republic of China. 2 Present address: ELEGI/Colt Laboratory, Centre for Inflammation Research, The Queen’s Medical Research Institute, University of Edinburgh, Edinburgh, Scotland. 1
0013-9351/$ - see front matter r 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.envres.2005.09.005
observed in epidemiological studies and which components of PM mediate such effects are still not clear. Although epidemiological and toxicological evidence suggests that it is the fine (PM2.5) and even the ultrafine (PM0.1) fractions that are responsible for the observed effects, there is no general agreement (Schins et al., 2004; Zhang et al., 2002). The wide range of endpoints suggest that more than one component may be driving the health effects (Dreher, 2000; Donaldson et al., 2003). Some leading hypotheses involve transition metal content and iron mobilization (Molinelli et al., 2002; Aust et al., 2002), particle size and surface (Donaldson et al., 2003), endotoxin contamination (Schins et al., 2004), or organic compounds such as polycyclic aromatic hydrocarbons (Nel et al., 2001). A remarkable consistency with regard to the adverse health effects of PM has been observed throughout many
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epidemiological studies irrespective of the locations where these studies have been carried out, i.e., with different PM composition. However, recent research has demonstrated that the adverse health outcomes vary from city to city and therefore indicate that specific components or properties of PM may be involved (Katsouyanni et al., 2001). Independent studies involving human volunteer exposures to PM indicate that PM-induced health effects may be due to its chemical composition and more specifically to its transition metal content (Ghio and Devlin, 2001; Schaumann et al., 2004). Transition metals are considered to exert their effects predominantly through the formation of hydroxyl radicals (dOH) via the Fenton reaction (Donaldson et al., 1997) and have been implicated in the inflammatory effects (Schaumann et al., 2004; Carter et al., 1997) and in the DNA damaging properties (Prahalad et al., 2001; Knaapen et al., 2002) of PM. We, along with others, have previously demonstrated that PM induces DNA strand breakage and formation of the hydroxyl-radical-specific lesion 8-hydroxy-20 -deoxyguanosine (8-OHdG) (Marnett, 2000) in lung epithelial cells (Prahalad et al., 2001; Knaapen et al., 2000, 2002; Dellinger et al., 2001; Brits et al., 2004). Furthermore, in a recent biomarker study involving Copenhagen students, personal exposure to PM was found to correlate with their lymphocyte 8-OHdG levels (Sorensen et al., 2003). Electron paramagnetic resonance (EPR), which is a wellestablished method to determine the radical generation properties of various dusts, had been applied by us previously to demonstrate that PM generates dOH in close relation with its ability to induce damage to naked DNA in suspension (Knaapen et al., 2002). More recently, we have shown that the dOH-generating properties of PM and its ability to induce oxidative damage in naked DNA varies considerably with sampling time (Shi et al., 2003a, b), but the implications of this variability with regard to biological effects in human lung cells remain to be elucidated. The aims of the present study were to determine the dOH-generating properties of coarse and fine PM collected over time at several contrasting locations within Germany and to relate these to different capacities to induce damage in naked DNA and in cellular DNA. 2. Materials and methods 2.1. Reagents 5,5-Dimethyl-1-pyrroline-N-oxide (DMPO), Hanks’ balanced salt solution (HBSS), ethidium bromide, Dulbecco’s modified Eagle’s medium (DMEM), phosphate-buffered saline (PBS), diaminobenzidine-tetrahydrochloride, desferoxamine, and dimethyl sulfoxide (DMSO) were obtained from Sigma–Aldrich (Taufkirchen, Germany). Hydrogen peroxide (H2O2) was purchased from Fluka (Seelze, Germany). Calf thymus DNA was obtained from Life Technologies Inc. (Gaithersburg, MD, USA). Vectastain-ABC kit was obtained from Vector Laboratories (Burlingame, CA, USA). For EPR experiments, double-distilled deionized water was used.
19
2.2. Collection and sample processing of particulate matter Coarse (2.5–10 mm) and fine (o2.5 mm) fractions of PM10 were sampled in weekly intervals at four locations in Nordrhein Westfalen, Germany in the period of February–May 2000. The locations were Borken (Bo) representing a rural site with approximately 40,000 inhabitants (265 inhabitants/km2) and three urbanized/industrialized sites in Dortmund (Do) with approximately 0.6 million inhabitants (population density 2100 inhabitants/km2) and Duisburg (Du-M and Du-B) with 0.5 million inhabitants (2200 inhabitants/km2). The geographical distances between Duisburg and Borken and between Duisburg and Dortmund are both approximately 50 km. Compared with Borken, both Duisburg and Dortmund are characterized by higher levels of NOx and CO. These locations are included in past and ongoing epidemiological studies on air quality and health (Polat et al., 2002). Coarse and fine PM were collected on preweighed Teflon filters using Graseby–Anderson dichotomous lowvolume samplers at a flow of 16.7 L/min. Filters were stored in the dark in a dry atmosphere until further analysis. The PM was removed from the filters by agitation (5 min) in 1 mL of ultrapure water, followed by sonication for 5 min. Resulting PM concentrations were immediately estimated using comparative turbidometry against a standard dilution curve using a carbon-black suspension. Although this method represents only an indirect estimate of the extracted amount of PM, it allowed for the analysis of freshly prepared (i.e., nonfrozen) PM suspensions. This approach avoids prolonged storage and freeze-thawing of PM suspensions, which can lead to altered leaching of metals or organic constituents and which also could affect particle agglomeration (Donaldson et al., 1997). Upon reconstitution of the filters from which the PM was extracted, the actual extracted mass was also determined gravimetrically, to allow a comparison with the turbidometry determinations. Particle suspensions were diluted in ultrapure water at the concentrations shown and immediately used for analysis of dOH generation, 8-OHdG formation, and DNA strand breakage experiments. Samples were all adjusted to equal concentrations, i.e., to 320 mg/mL for coarse PM and 380 mg/mL for fine PM. For determination of the regional variability of dOH and 8-OHdG formation, pairs of fine and coarse PM were analyzed randomly with regard to sampling period and sampling location. A random selection of fine PM was also used for the determination of DNA strand breaks in A549 human lung epithelial cells.
2.3. Electron paramagnetic resonance Hydroxyl radical formation by the coarse and fine PM was evaluated by EPR as described previously (Shi et al., 2003a, b). Briefly, 50 mL of the freshly prepared particle suspension was mixed with 100 mL of the spin trap DMPO (0.05 M in distilled deionized water) and 50 mL H2O2 (0.5 M in PBS). The suspension was incubated for 10 min at 37 1C in a shaking water bath and filtered through a 0.1 mm filter (Acrodisc 25 mm syringe filter, Pall Gelman Laboratory, Ann Arbor, MT, USA). The filtrate was immediately transferred to a capillary and measured with a Miniscope EPR spectrometer (Magnettech). The EPR spectra were recorded at room temperature using the following instrumental conditions: magnetic field, 3360 G; sweep width, 100 G; scan time, 30 s; number of scans, 3; modulation amplitude, 1.975 G; receiver gain, 1000. Quantification was done by accumulation of three spectra, each averaging three scans. All four peaks were quantified by measuring the amplitudes, and outcomes are expressed as the total amplitude in arbitrary units (AU). Comparative evaluation of this quantification protocol with the method of double integration as usually applied in EPR studies demonstrated nearly identical results for DMPO-OH spectra (n ¼ 30, Pearson’s r ¼ 0:99, Po0:001).
2.4. Hydroxydeoxyguanosine induction by PM in calf thymus DNA Induction of the dOH-specific DNA lesion 8-hydroxydeoxyguanosine by PM in isolated calf thymus DNA was estimated via a dot-blot assay
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that we have developed recently (Knaapen et al., 2002) based on a method described by Musarrat and Wani (1994). Freshly prepared suspensions of PM were incubated with 50 mg of calf thymus DNA dissolved in Tris–HCl (10 mM, pH 8.0) and H2O2 (1 mM). In each experiment, DNA incubated without PM and H2O2, and DNA incubated with 0.1 mM FeSO4 and 1 mM H2O2 were included, respectively, as negative and positive controls. Samples were incubated in the dark for 90 min at 37 1C in a shaking water bath and then immediately centrifuged (6000 rpm, 5 min). Then 200 mL of the supernatant was transferred to a fresh tube, and DNA was precipitated by the addition of 1/10 vol. NaAc (1.5 M, pH 6.0) and 2 vols 100% icecold ethanol at 20 1C for 1 h. The DNA was then washed twice using 70% ethanol (13,000 rpm, 5 min), dried in the dark, dissolved in 30 mL of Tris–HCl buffer, and stored overnight at 4 1C. DNA concentrations were determined spectrophotometrically and the samples were diluted to a final concentration of 2.56 mg/mL in 20 standard saline citrate. Of each sample, replicate two-fold dilutions were blotted on a nitrocellulose membrane using a dot-blot apparatus. To each blot, both negative and positive controls were added. The DNA was cross-linked by baking the membrane for 90 min in a prewarmed oven at 80 1C. Blocking of the membrane was performed overnight using casein. Immuno-localization of 8-OHdG was performed using the N45.1 monoclonal antibody (Toyokuni et al., 1997) and the Vectorstain-ABC kit with diaminobenzidine staining according to the recommended protocol (Vector Laboratories). The blots were analyzed by computer-assisted densitometry scanning (Bio-Rad) and expressed relative to the density of the negative controls. The relative staining intensities as ranked for each separate dot-blot experiment were used for statistical evaluation.
2.5. Treatment of A549 cells with fine PM A549 cells (American Type Culture Collection) were grown in DMEM supplemented with 10% heat-inactivated fetal calf serum, L-glutamine, and 30 IU/mL penicillin–streptomycin at 37 1C and 5% CO2. For experiments, cells were trypsinized at confluence, seeded into 60-mm culture dishes, and grown until confluence. Cells were washed two times with HBSS and then treated with freshly prepared samples of fine PM as described before upon dilution in HBSS at a final concentration of 20 mg/ cm2. Cells were incubated for 3 h at 37 1C (100% relative humidity, 5% CO2). A total of 15 fine PM samples were randomly selected three independent experiments measuring each sample in duplicate. In additional experiments, DMSO and DMPO were used as hydroxyl radical scavengers.
2.6. DNA strand break analysis DNA strand break formation in A549 cells was determined by the alkaline comet assay (Tice et al., 2000), as described previously (Schins et al., 2002). Fully frosted slides were covered with a layer of 0.65% agarose using a cover slide and stored overnight at 4 1C. Following treatment, A549 cells were harvested from the dishes using trypsin and were then suspended in HBSS. Cytotoxicity in A549 cells caused by exposure procedures and cell processing was evaluated using Trypan blue dye exclusion. Subsequently, 25 ml of the cell suspension (approximately 2 106 cells/mL) was mixed with 75 mL 0.5% low-melting-point agarose. This mixture was then added to the slides on top of the first agarose layer using a cover glass. Slides were stored 45 min at 4 1C to allow solidification and covered with another layer of low-melting-point agarose (100 mL). Following solidification for at least 45 min at 4 1C, slides were immersed in lysis buffer (2.5 M NaCl, 100 mM EDTA, 10 mM Tris base, 1% sodium lauryl sarcosinate, pH 10, 10% DMSO, and 1% Triton X-100 added just before use) and stored overnight at 4 1C. The following day, slides were rinsed with distilled water and placed in an electrophoresis tank filled with ice-cold buffer (300 mM NaOH, 1 mM EDTA, pH 13) for 30 min. Electrophoresis was conducted at 300 mA and 25 V for 15 min. Slides were then neutralized 3 10 min using neutralization buffer (0.4 M Tris, pH 7.5). All steps described were performed in the dark or under dimmed red light to prevent additional DNA damage. Slides were stained with
ethidium bromide (20 mg/mL in H2O) and comet appearances were analyzed using an Olympus BX60 fluorescence microscope at 1000 magnification. On every single slide 50 cells were analyzed randomly and classified into one of five categories according to tail length (0, 1, 2, 3, 4, in which 0 ¼ no tail). For final analysis a comet score of each individual slide was calculated according to the method described in Knaapen et al. (2002): comet score ¼ sum (class 1 cells+2x class 2 cells+3x class 3 cells+4x class 4 cells). Using this formula, a minimally damaged sample will have a score of 0, whereas a maximally damaged sample obtains a comet score of 200. In further experiments, comet appearances were also evaluated using the comet image analysis software program Comet Assay II (Perceptive Instruments, Haverhill, UK).
2.7. Statistical analysis Comparisons between fine and the coarse PM or of the PM samples of different sampling sites (rural area (Bo) versus other sites) were carried out using the t-test or the nonparametric Mann–Whitney u test (for 8-OHdG only). All correlation was determined using simple linear regression analysis.
3. Results The mean masses of the PM sampled at the different locations and extracted from the Teflon filters using our protocol are summarized in Table 1. The sampled masses and the extracted masses were remarkably similar for the four locations, with the exception of coarse PM from Dortmund, which was significantly higher than coarse PM from Borken. Using our extraction protocol (vortexing and sonication) the mass recovered from the Teflon filters appeared to be quite high for the coarse fraction (mean: 97%; range: 84–105%) and the fine fraction (mean: 89%; range: 86–92%). For both PM size fractions, the recovery appeared to be a constant proportion of the filter loading as indicated by a linear correlation between the sampled and the recovered mass (fine: r ¼ 0:92, Po0:001; coarse: r ¼ 0:69, Po0:001). Since turbidometry is the only method that can be used to estimate mass for immediate use with small amounts of PM, we analyzed the relationship between gravimetric and turbidometric data and found a highly significant correlation between mass and density for both the fine and coarse fractions. Similar correlation coefficients were found for the fine (r ¼ 0:58, n ¼ 40, Po0:01) and the coarse (r ¼ 0:41, n ¼ 41, Po0:01) fractions, but the slope of the line shows that, in our hands, the concentrations expressed by turbidometry lead to an underestimation (30%) of extracted mass. Since hydroxyl radicals produced via Fenton-like reactions have been considered key features of PM, we used EPR with spin-trapping to quantify dOH formation by PM as an entirety. All PM samples collected during this study showed the typical spectrum for DMPO-OH, differing only in signal amplitude of dOH formation. The quantification of the EPR measurements is shown in Fig. 1. As can be seen in the figure, urban PM had significantly higher dOH generation capacity than rural PM. The coarse fractions generally showed a higher signal than the fine
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Table 1 Characterisation of fine (1a) and coarse (1b) PM sampled at four different locations Borken
Dortmund
Duisburg-meid
Fine PM Number of samples Sampled mass (mg) Extracted mass (mg) Recovery (%) Estimation of extracted PM (mg/mL) (turbidometry)
12 2.270.6 2.070.6 88.9711.6 0.570.1
7 2.770.7 2.570.6 92.374.8 1.070.4
Coarse PM Number of samples Sampled mass (mg) Extracted mass (mg) Recovery (%) Estimation of extracted PM (mg/mL) (turbidometry)
14 1.870.5 1.670.5 93.5714.3 0.570.2
7 2.771.0 b 2.770.9 b 98.377.7 0.670.1
Duisburg-bus
14 2.370.8 2.170.8 89.4724.5 0.670.2
7 2.170.7 1.870.7 86.2710.3 0.670.2
14 2.371.1 2.070.6 105.1746 0.670.3
6 2.371.0 1.870.7 84.1729.3 0.670.2
160
EPR Signal (A.U)
140
fine Coarse
* **
120 100
**
**
80
EPR Signal (A.U)
All data are expressed as mean7standard deviation.
*
60
180 160 140 120 100 80 60 40 20 0 8
40
9
10
11
12
13
14
15
16
17
18
19
20
21
18
19
20
21
Sampling Weeks
(A)
0
Bo
Do
Du-M
Du-B
Fig. 1. Hydroxyl radical generation of coarse and fine PM of suspensions sampled at four locations. Data are expressed mean and SD of the intensity of the resulting DMPO-OH signal in arbitrary units from the weekly samples of coarse and fine PM. PM samples from the urban/ industrial areas (Do, Du-M, Du-B) showed higher ability to generate hydroxyl radical than the PM samples from the rural location (Bo). The asterisks indicate statistically significant differences from the rural location (Bo), at Po0:05 and Po0:01.
fractions, with the exception of samples from Dortmund. In addition to differences in both PM size and sampling location, considerable temporal variability in dOH formation was observed. Fig. 2 shows, as an example, the dOH generation for consecutive sampling weeks of both fine and coarse PM from the rural location (Bo) and an urban location (Du-M). As can be seen in the figure, irrespective of the week-to-week variations of the dOH signals, coarse PM consistently showed higher ability to generate dOH than fine PM for these locations. To evaluate whether the observed variability in dOH generation relates to the induction of 8-OHdG, coarse and fine fractions of PM were incubated with calf thymus DNA in the presence of 1 mM H2O2 and analyzed using an immuno-dot-blot assay. Both fine and coarse PM caused formation of 8-OHdG and the effects tended to be stronger for the PM samples collected at the urban/industrialized locations (Fig. 3). However, significantly higher 8-OHdG
EPR Signal (A.U)
20
(B)
180 160 140 120 100 80 60 40 20 0
8
9
10
11
12
13
14
15
16
17
Sampling Weeks
Fig. 2. Time course of week means of DMPO-OH formation of the fine (A) and the coarse (B) fraction of PM as sampled in the rural town (Bo) (m) and a location of the city of Duisburg (Du-M) (’). Data are expressed as the intensity of the resulting DMPO-OH signal in arbitrary units. PM sampled at the urban area cause higher ability to generate hydroxyl radical. Variations were observed for both fractions and locations, large variations were found among urban samples.
was observed only with coarse PM from Duisburg-M compared to coarse PM from the rural location Borken. A significant correlation was found between the dOH generation as determined by EPR and the induction of 8OHdG (n ¼ 81, Spearman’s r ¼ 0:48, Po0:001). To determine the significance of these observations for cellular DNA damage, A549 cells were treated with fine PM samples, randomly selected from both urban and rural locations. DNA strand breakage results are presented in Fig. 4. As can be seen in Fig. 4A, no significant differences between the different sampling locations were found in DNA damage, but a considerable variability among the
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5
3 2
4 3
1
1
0
0 Do
Du-M Du-B
40 20 0
Bo
Do Du-M Du-B
DNA damage (comet score)
DNA damage (comet score)
(A)
60
(B)
10
#
##
PM & DMPO
PM & DMSO
#
5 0 control
PM
DMPO
DMSO
3 ** Bo
Do
Du-M Du-B
Fig. 3. Induction of 8-OHdG by fine and coarse fractions of PM in calf thymus DNA. The data shown are ranked 8-OHdG in accordance with the relative staining intensities for each separate experiment (ranks 1–6). For each sampling location the horizontal line represents the median value. Urban area PM demonstrated a higher ability to induce 8-OHdG than rural PM. Significant differences were observed only in urban PM (Du-M) (Po0:05) compared to rural particles (Bo).
80
**
2
tail moment
4
tail intensity (%)
5
Bo
15
Coarse PM 6
8-OHdG (rank)
8-OHdG (rank)
Fine PM 6
2 ## 1
##
#
##
0 control
PM
PM & DMPO
PM & DMSO
DMPO
DMSO
Fig. 5. DNA strand breakage by fine PM in the presence or absence of the hydroxyl radical scavengers DMPO and DMSO. A549 cells were treated for 3 h with PM suspended in HBSS (300 mg/mL) and in the presence or absence of DMPO (15 mM) or DMSO (0.5% in v/v). DNA damage as analyzed by a comet image analysis software program are expressed as % tail DNA (A), and tail moment (B) (data are mean and standard deviation; n ¼ 4). Both scavengers caused significant inhibition of PMinduced DNA strand breakage, whereas the scavengers alone had no effect on DNA strand breakage ( Po0:01 versus control; ]Po0:05 versus PM; ]]Po0:01 versus PM).
80 60 40 20 0 20
40
60
80
DMPO-OH (A.U)
Fig. 4. Induction of DNA strand breakage by fine PM from different sampling locations in A549 cells (A) and its relation with hydroxyl generation (B). A549 cells were treated for 3 h with fine PM samples from different locations at equal doses (20 mg/cm2). No significant differences in DNA damage were found in the different sampling locations (A). When samples were subdivided into rural and urban/industrial locations (B), different slopes were observed in the hydroxyl-radical-generating ability of the samples as determined by EPR and the induction of DNA strand breakage in A549 cells, respectively, for rural samples (J) versus urban samples (K).
samples, in line with the dOH-generating properties of the different samples, was observed. In Fig. 4B, the dOHgenerating capacities of the PM samples tended to correlate with their ability to elicit DNA strand breakage in the A549 cells. When samples were subdivided into rural and urban samples, clear associations between dOH generation and DNA strand breakage were observed albeit with different slopes. To evaluate whether dOH generation, as determined by EPR, may indeed be involved in the observed DNAdamaging effects of PM, subsequent experiments using the hydroxyl-radical scavengers DMSO and DMPO were performed. The effects of DMSO and DMPO on DNA strand breakage by fine PM are shown in Fig. 5. Here, coincubation with both DMSO and DMPO caused significant reductions of DNA strand breakage in the A549 cells.
4. Discussion The data in our present study are in line with earlier observations that PM can elicit DNA damage in lung epithelial cells via inherent oxidative properties. It is generally accepted that reactive oxygen species (ROS) are able to cause DNA strand breaks and DNA oxidation, processes that have been implicated in the initiation stage of carcinogenesis (Marnett, 2000). Among the major products of oxidative DNA damage is the premutagenic lesion 8-hydroxy-20 -deoxyguanosine (Marnett, 2000; Collins et al., 1993). Indeed, a clear correlation between hydroxyl-radical generation by PM and induction of 8OHdG in naked DNA was observed in our study, which was also in line with our previous observations (Shi et al., 2003a, b). Obviously and in contrast to acellular methods such as EPR and naked DNA damage assays, the oxidative DNAdamaging properties of PM in cellular systems involves more complex biochemical and physical processes, where various constituents of PM may interact with components on the surface and inside the cell. The EPR method and the 8-OHdG assays employed in this research are both performed in the presence of exogenous hydrogen peroxide and therefore most likely reflect the Fenton reactivity of the samples. Several other mechanisms have been considered, e.g., generation of ROS such as hydroxyl radical by PM as a result from its surface area (Kuchino et al., 1987) and
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organic constituents (Nel et al., 2001; Brown et al., 2001). It has been found that dOH generation may also be modified by other organic and inorganic components, such as sulfates (Li et al., 2002) and semiquinones (Dellinger et al., 2001). Previously, we and others have shown that PM induces both DNA strand breaks and 8-OHdG in lung epithelial cells (Prahalad et al., 2001; Knaapen et al., 2000, 2002; Dellinger et al., 2001; Brits et al., 2004; Ghio et al., 1999). Inhibition of PM-induced DNA strand breakage with hydroxyl-radical scavengers such as tetramethylthiourea or dimethyl sulfoxide, the antioxidant enzyme catalase, and the iron chelator deferoxamine suggest that dOH produced by the Fenton reaction may play a predominant role in these cellular systems (Knaapen et al., 2002; Dellinger et al., 2001). The data from our present study support these observations, since for fine PM a correlation between DNA damage in A549 cells and hydroxyl-radical generation within this PM fraction was observed. Furthermore, it was found that the hydroxylradical scavengers DMPO and DMSO were capable of inhibiting strand breakage by fine PM. With regard to the current effects as observed with fine PM, we have recently demonstrated, using immunocytochemistry, that this fraction and coarse PM elicit 8-OHdG formation in A549 cells (Shi et al., 2003a, b). However, in that study we were not able to correlate dOH generation to this oxidative DNA lesion, due to the semiquantitative nature of the method. Quantitative analysis of 8-OHdG, e.g., using high-performance liquid chromatography with electrochemical detection (Don Porto Carero et al., 2001), necessitates incubations of large cell numbers to obtain sufficient DNA and therefore high mass of PM. In this respect, a more useful approach for the quantitative detection of DNA damage, i.e., the alkaline comet assay which requires relatively low cell numbers for analysis (Tice et al., 2000) was used and this was suitable in our hands given the limited collected amount of PM in our setup. Recently developed modifications of the comet assay, which, for example, allow specific identification of oxidative lesions such as 8-OHdG (Schins et al., 1995), were unfortunately not routine in our lab at the time of measurement. Nevertheless, taken together, our previous studies with 8OHdG (Shi et al., 2003a, b) and our current observations on DNA strand breakage in relation to EPR measurements and the effect of DMSO and DMPO indicate that the comet assay represents an indicator of oxidative stress by PM in A549 cells. Importantly, we showed that PM elicits DNA strand breakage in cultured cells in the absence of exogenous hydrogen peroxide, whereas hydroxyl-radical generation by PM using EPR has been measured upon addition of a rather high concentration of H2O2. Previously, we showed that micromolar levels of H2O2, concentrations occurring in physiological conditions, already enhance hydroxyl radical generation by PM (Knaapen et al., 2002; Shi et al., 2003a, b). Complementary to our EPR work, EPR measurements of dry PM samples also show spectra
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indicative of semiquinone radicals similar to those found in cigarette tar samples, suggesting that H2O2 formation via quinone cycling (Dellinger et al., 2001) can attribute to dOH generation. Our data demonstrate that, although PM samples from different areas have the ability to generate hydroxyl radicals and cause DNA damage, the patterns may differ between sampling locations. On the one hand, PM samples from an industrial/urban location showed higher ability for the formation of hydroxyl radicals than rural PM compared at equal mass. On the other hand, no clear differences were found in the induction of 8-OHdG in naked DNA or DNA strand breakage in lung epithelial cells by PM samples from different locations. Despite these observations, for both rural and urban samples, DNA damage appeared to increase with increasing hydroxylradical-generating capacities. Obviously, however, it should be emphasized that our results are observed with a relatively low number of PM samples and these results should be interpreted with some caution. Therefore further studies are needed to confirm the contribution of hydroxylradical generation toward the induction of DNA damage. In conclusion, we have demonstrated that PM, both coarse and fine, sampled over time and at different locations have the ability to generate hydroxyl radicals, induce 8-OHdG formation in calf thymus DNA, and elicit DNA strand breaks in human lung epithelial cells. Importantly, PM shows considerable variability in each of these endpoints with regard to the sampling location and time. DNA strand breakage appeared to correlate with the hydroxyl-radical-generating capacities of the PM samples but with different profiles for rural versus urban/industrial samples. The observed location-specific characteristics of PM are in agreement with recent observations in epidemiological research (Katsouyanni et al., 2001) and forward a message that geographic or site-related physical–chemical characteristics of PM impact its ability to elicit cellular oxidative stress and DNA damage via mechanisms involving dOH generation.
Acknowledgments The PM samples used for this study have been collected within the framework of the so-called HOTSPOT study, a health surveillance program in Nordrhein Westfalen, supported by the Landesumweltamt (LUA) in Essen, Germany. We acknowledge Ms. Olschewski and Mr. Schwarz from the LUA for their assistance in PM sampling.
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