Subway Particles Are More Genotoxic than Street Particles and Induce

Chem. Res. Toxicol. 2005, 18, 19-23

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Subway Particles Are More Genotoxic than Street Particles and Induce Oxidative Stress in Cultured Human Lung Cells Hanna L. Karlsson,† Lennart Nilsson,‡ and Lennart Mo¨ller*,† Unit for Analytical Toxicology, Department of Biosciences, Karolinska Institutet, SE-141 57 Huddinge, Stockholm, Sweden, and Department of Neuroscience, Karolinska Institutet, SE-171 77, Stockholm, Sweden Received October 5, 2004

Epidemiological studies have shown an association between airborne particles and a wide range of adverse health effects. The mechanisms behind these effects include oxidative stress and inflammation. Even though traffic gives rise to high levels of particles in the urban air, people are exposed to even higher levels in the subway. However, there is a lack of knowledge regarding how particles from different urban subenvironments differ in toxicity. The main aim of the present study was to compare the ability of particles from a subway station and a nearby very busy urban street, respectively, to damage DNA and to induce oxidative stress. Cultured human lung cells (A549) were exposed to particles, DNA damage was analyzed using single cell gel electrophoresis (the comet assay), and the ability to induce oxidative stress was measured as 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) formation in lung cell DNA. We found that the subway particles were approximately eight times more genotoxic and four times more likely to cause oxidative stress in the lung cells. When the particles, water extracts from the particles, or particles treated with the metal chelator deferoxamine mesylate were incubated with 2′-deoxyguanosine (dG) and 8-oxodG was analyzed, we found that the oxidative capacity of the subway particles was due to redox active solid metals. Furthermore, analysis of the atomic composition showed that the subway particles to a dominating degree (atomic %) consisted of iron, mainly in the form of magnetite (Fe3O4). By using electron microscopy, the interaction between the particles and the lung cells was shown. The in vitro reactivity of the subway particles in combination with the high particle levels in subway systems give cause of concern due to the high number of people that are exposed to subway particles on a daily basis. To what extent the subway particles cause health effects in humans needs to be further evaluated.

Introduction The adverse health effects of airborne particles and the mechanisms underlying these effects are under scientific debate. Particulate matter (PM)1 is a complex mixture that often is divided in fractions mainly depending on size. Particles with an aerodynamic diameter less than 10 µm (PM10) are small enough to reach conductive airways and the lower respiratory system. Epidemiological studies show that particles lead to a number of health problems including respiratory and cardiovascular diseases, aggravated symptoms for asthmatics, adverse effects on lung development in children, and excess deaths in the population (1-4). Urban particles are also associated with an increased risk of lung cancer (5), and recent data suggest heritable genetic changes in mice (6). The dominant mechanistic hypothesis for particulate-induced health effects is oxidative stress and inflammation (7, 8). Oxidative stress occurs when antioxi* To whom correspondence should be addressed. Tel: +46 8 608 91 89. Fax: +46 8 774 68 33. E-mail: [email protected]. † Department of Biosciences. ‡ Department of Neuroscience. 1Abbreviations: PM, particulate matter; ROS, reactive oxygen species; 8-oxodG, 8-oxo-7,8-dihydro-2′-deoxyguanosine; dG, 2′-deoxyguanosine; GTC, guanidine thiocyanate; XRD, X-ray diffraction.

dant systems are overwhelmed by oxidative processes and can result from particles via production of metalgenerated reactive oxygen species (ROS) (9), redox cycling by semiquinone radicals from organic compounds adsorbed on the particles (10), and from ROS produced by activated macrophages. Oxidative stress can lead to up regulation of inflammatory cytokines as well as damage to macromolecules and is believed to be a mechanism behind several diseases and aging (11). The oxidative DNA lesion 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) is promutagenic (12) and is used as a biomarker for oxidative stress. In rats chronically exposed to particles, 8-oxodG was increased at an early stage in the lung tissue of animals that later developed lung cancer (13). The relevance of rat models has however been questioned due to the fact that tumors in rats are seen when particle deposition overwhelms the clearance mechanisms of the lung resulting in “overload” (14) followed by chronic inflammation, cell proliferation, and secondarily oxidative stress and DNA damage. However, an association between personal exposure to particles less than 2.5 µm (PM2.5) and 8-oxodG in lymphocyte DNA has been found (15) as well as an association between PM2.5 exposure and urinary 8-oxodG in workers exposed to particles (16).

10.1021/tx049723c CCC: $30.25 © 2005 American Chemical Society Published on Web 12/24/2004

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Although urban street level concentrations of particles are high, the daily exposure in many large cities may depend even more on exposure from the subway system where particle levels can be in the range of 500-1000 µg/m3 as recently reported in Stockholm (17) and London (18, 19). The subway particles are different in composition as compared to those arising from combustion, and little is known about their toxic effects. The main aim of the present study was to compare the ability of particles from a subway station and a nearby urban street, respectively, to damage DNA and to induce oxidative stress. Cultured human lung cells (A549) were exposed to particles, DNA damage was analyzed using single cell gel electrophoresis (the comet assay), and the ability to induce oxidative damage was measured as 8-oxodG formation in lung cells and in a 2′-deoxyguanosine (dG) solution, using HPLC with electrochemical detection. Furthermore, interactions between the different particles and the lung cells were visualized using electron microscopy.

Materials and Methods Particle Collection. Particles were collected using two different methods: (i) A sampler similar to the so-called Gent filter unit was used for low-volume sampling of PM10 on a 47 mm (Zefluor 2.0 µm) Teflon filter equipped with an open-faced NILU Filter Holder. Daily samples (24 h) were collected at a flow rate of 17 L/min. (ii) The high-volume sampler SierraAndersen/GMW model 1200 was used to collect PM10 particles on a glass fiber filter. Daily samples were collected at a flow rate of about 390 L/min. Preparation of Particles. The support ring from the Teflon filter was cut, and the filter was weighed and immersed in 0.5 mL of MilliQ water in a glass tube. The tube was vortexed (10 min) and sonicated (15 min) in a water bath subsequently two times where after it was immersed in another 0.5 mL of MilliQ water, vortexed, and sonicated again. The particle suspensions were pooled, and the filter was dried in room temperature overnight. Particles from the glass fiber filters were removed more “soft” in order to prevent breakage of the filter. A piece (about 2 cm × 2 cm) was cut from the filter, weighed, and immersed in 1 mL of MilliQ water in a glass tube. The tube was gently shaken by hand for 2 min followed by sonication in a water bath for 5 min. The filters were dried in a SpeedVac overnight. Blank filters were treated the same way and were used as controls in all experiments. The concentrations of particles in the water suspensions were based on the difference in filter weighs before and after particle removal. Blank filter extractions showed no measurable weigh loss from the Teflon filters and 0.5-2% weigh loss from the glass fiber filters. All particle suspensions were used “fresh” within 1-3 days after preparation (stored in refrigerator) and were vortexed (5 min) and sonicated (15 min) prior to cell exposure. Exposure of Human Lung Epithelial Cells. A549 type II lung cells originally obtained from the American Tissue Type Collection (kindly provided by Prof. Ian Cotgreave) were grown in DMEM medium, supplemented with 10% fetal calf serum, 100 U/mL penicillin, 10 µg/mL streptomycin, and 1 mM sodium pyruvate in a humidified atmosphere at 37 °C and 5% CO2. Cells were seeded into 24 well plates (for comet assay) or 100 mm plates (for 8-oxodG analysis) and grown to 90-100% confluence for about 24 h. The particle suspensions were mixed with fresh medium, and cells were exposed for 4 h to 5, 10, 20, or 40 µg/cm2 (9-70 µg/mL) for the comet assay and 10 µg/cm2 (50 µg/mL) for 8-oxodG analysis. Noncytotoxic concentrations, determined by trypan blue staining, were used. Comet Assay. The alkaline version of the comet assay according to Singh et al. (20) was used with some modications. After exposure, the cells were washed with PBS (0.01 M, pH 7.4), trypsinisated, and suspended in cell medium. The suspen-

Communications sions were centrifuged at 200g for 5 min (4 °C), and the cells were washed with PBS. The cell pellet was suspended in 50 µL of PBS, and 12 µL was mixed with 70 µL (38 °C) of low melting point agarose (0.75% w/v), which was spread over a precoated (0.3% agarose) slide. The slides were put vertically in cold lysis buffer (1% Triton X-100, 2.5 M NaCl, 10 mM Tris, and 0.1 M EDTA, pH 10) for 1 h on ice and then in PBS containing 2 mM EDTA for 10 min. DNA unwinding was performed in cold alkaline solution (0.3 M NaOH and 1 mM EDTA) for 40 min followed by electrophoresis at 25 V (0.86 V/cm) for 30 min in a Sub-Cell GT unit containing the same alkaline solution. The slides were neutralized in 0.4 M Tris (pH 7.4) for 5 min twice and then in water for 5 min. They were dried overnight in room temperature and fixed in methanol for 5 min. After they were stained with ethidium bromide, the comets were examined with a BH-2 fluorescence microscope with a 20× apochromatic objective, using the program Komet 4.0 (Kinetic Imaging Ltd, Liverpool, United Kingdom). Thirty-five comets each on two different slides were evaluated for each sample at each experiment. dG Reaction. dG (400 µM) in 0.1 M potassium phosphate buffer, pH 7.4, was reacted with (a) 0.4 mg/mL particles, (b) 0.4 mg/mL particles that had been pretreated with 0.5 mM deferoxamine mesylate for 30 min (0.2 mM final concentration in dG reaction), (c) water extracts from 0.4 mg/mL particles, or (d) 0.4 mg/mL particles in combination with 0.1 mM H2O2. The water extracts were prepared by centrifuging the particle suspensions at 14000g for 2 min followed by filtration of the supernatant through a Nanosep (30K) filter. The reaction mix was incubated at 37 °C for 30 min and was mixed every 10 min during incubation. The reacted samples were immediately frozen at -80 °C. Analysis of 8-OxodG with HPLC-EC. A cold (0 °C) high salt GTC (4 M guanidine thiocyanate) DNA extraction method was used (21). All solutions were chelex treated to remove transition metals, and all steps except DNA hydrolysis were performed at 0 °C. The HPLC system, which is described in more detail elsewhere (21), consisted of an isocratic Scantec pump set at 0.75 mL/min, a C18 Opti-Guard column (15 mm × 1 mm i.d., Optimize, Portland, OR), and two Delta-Pak (150 mm × 3.9 mm i.d., 5 µm) reversed phase columns (Waters, Milford, MA). 8-OxodG was detected with an electrochemical detector (Coulochem II, ESA, Chelmsford, MA) with a graphite filter protected 5011 analytical cell (ESA; screen electrode, +200 mV; analytical electrode, +350 mV). dG was detected with a 486 UV detector (Waters) set at 290 nm. The HPLC buffer consisted of 10% v/v methanol and 20 mM sodium acetate set to pH 5.3 with acetic acid. Calibration curves for dG and 8-oxodG were made from standards every day before analysis. The total HPLC run time was 20 min, and the retention times for dG and 8-oxodG were approximately 12 and 16 min, respectively. Analysis of Particle Composition. Analysis of the atomic composition of a single particle was performed by means of scanning electron microscopy and X-ray energy dispersive spectrometry. Crystalline compounds were analyzed in the subway and street samples by using X-ray diffraction (XRD) analysis. The particles on the filters were analyzed using a Philips XRD with Cu KR X-ray tube. Image Analysis. A549 cells were seeded on small microscopic slides in chambers and grown to 90-100% confluence for about 24 h. The particle suspensions were mixed with fresh medium, and cells were exposed for 1 h. The cell medium was removed, and the cells were washed three times and fixed in 3% glutaraldehyde. The images were captured by a JSM-840 scanning electron microscope.

Results DNA Damage Analyzed with the Comet Assay. Both types of particles induced DNA damage in a concentration-dependent manner (Figure 1), but the subway particles were more potent at each concentration. The slope of the concentration-response curve was

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Figure 1. DNA damage in cultured human lung cells after exposure to particles from the subway ([) or from the urban street (0), respectively. Each point represents the mean of four independent experiments ( SD. The subway particles differ significantly (p < 0.01) from control at all concentrations from 10 µg/cm2 and the street particles from 20 µg/cm2.

almost eight times larger for the subway particles. Particles from the subway formed significantly (t-test, p < 0.01) higher levels than the controls at all concentrations from 10 µg/cm2 and the street particles from 20 µg/cm2 (p < 0.01). 8-OxodG Formation in A549 Cells. Analysis of 8-oxodG in A549 cells showed low levels (0.7 ( 0.4 8-oxodG/106 dG) in cells exposed only to water extracts from the blank filter (Figure 2a). No statistical increase was seen in cells exposed to 10 µg/cm2 street particles, but the same concentration of subway particles showed four times higher levels (2.8 ( 0.6 8-oxodG/106 dG, p < 0.01). Oxidation of dG in Solution. The subway particles showed a higher oxidative capacity than the street particles also when incubated with dG (Figure 2b). Pretreatment of the particles with the metal chelator deferoxamine mesylate prevented around 30% of the oxidation from the street particles and 80% of the oxidation from the subway particles, indicating that metals were involved in the oxidation for both types of particles but especially for the subway particles. The oxidative capacity of the water extracts from the particles showed that over half of the oxidation (58%) from the street particles was due to soluble substances. The corresponding value for the subway particles was only 7% indicating that most of the oxidation was due to nonsoluble, redox active substances. The oxidation increased dramatically (8-10 times) when 0.1 mM H2O2 was added in combination with the particles but only slightly (1.4 times) when 0.1 mM H2O2 was added to the control (Figure 2c). Elemental Composition. Analysis of the atomic composition of a typical subway particle by means of scanning electron microscopy showed that oxygen and iron dominated the mass of the particle (Figure 3). Furthermore, analysis of crystalline compounds by XRD indicated that iron present in the street particles was mainly in the form of hematite (Fe2O3) whereas Fe from the subway was mainly magnetite (Fe3O4). Image Analysis. Scanning electron images of particles and lung cells, which were captured after the cells had been washed several times, revealed that particles were attached to the cells (Figure 4). The urban street particles showed a typical ball-like shape with rough

Figure 2. (a) 8-OxodG in cultured human lung cells after exposure to 10µg/cm2 particles from urban street and subway, respectively. The bars represent means of four independent experiments ( SD (**p < 0.01). (b) Increase in oxidation of dG as compared to control after incubation with (1) particles (black), (2) particles treated with the metal chelator desferoxamine (gray), or (3) water extract from particles (white) (**p < 0.01). (c) Oxidation of dG after incubation with particles (black) or particles in combination with H2O2 (gray) (***p < 0.001, as compared to without H2O2). The addition of H2O2 led to 8-10 times and 1.4 times increased oxidation for the particles and the control, respectively.

Figure 3. Atomic composition (%) of a typical subway particle measured by scanning electron microscopy and X-ray energy dispersive spectrometry.

surfaces often in clusters, which is to be expected from particles generated from combustion. The subway particles were different. A common structure was one flat surface, in some cases in combination with parallel scratches and sharp edges. Other subway particles looked like metal sheets or flakes. Parts of some particles were

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Figure 4. Electron microscopy images of human lung cells (red) exposed to particles. Panels A-C represent subway particles, and panel D represents particles from the urban street. The particles seen are attached to cells and have an approximate width of 6 (A,B), 4 (major particle, C), and 1 (circular particle close to the middle section of the image, D) µm. The atomic composition analysis shown in Figure 3 was done on a typical subway particle with one flat surface.

over grown by cell structures, or the particle itself was attached to the cell as an arrowhead penetrating the cell membrane.

Discussion There is an increasing interest in the health effects of particles and what properties of the particles that contribute most to their toxicity. This study showed a striking difference between PM10 from the subway and street respectively, regarding genotoxicity measured with the comet assay. The subway particles were more genotoxic, and furthermore, they also induced oxidative stress in cultured human lung cells. It is likely that also the street particles would cause oxidation in cultured cells at higher concentrations. Prahalad et al. (22) showed that urban dust particles from Washington, DC, and Du¨sseldorf (Germany) caused oxidation of DNA in BEAS cells. The exposure was, however, much higher than in the present study. The dG experiment revealed that the oxidative capacity of the subway particles most likely was due mainly to redox active solid metal particles. The insoluble particle fraction of the subway particles was clearly redox active since almost no oxidation was seen when dG was reacted with the water extract. Other studies have shown that the insoluble particle core of different types of particles can be involved in redox reactions (23), contribute to DNA damage (24), or be responsible for induction of inflammatory mediators (25). Numerous experimental studies show, however, that the content of soluble metals is important for, e.g., genotoxicity (22) and inflammatory response (26) from particles. The decreased oxidative potential of subway particles seen after treatment with deferoxamine mesylate is likely due to binding of the chelator to the metal surface leading to prevention of surface iron to participate in redox reactions, which has been seen in studies involving asbestos (27).

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Analysis of the atomic composition of a typical subway particle by means of scanning electron microscopy showed that oxygen and iron dominated the mass of the particle (Figure 3). Iron is often the dominant metal also in ambient urban PM but is usually at levels around 1-4% of the mass (28) as compared to over 40% (w/w) in subway particles. The iron-rich particles are the most abundant particles also in the London Underground (19). Furthermore, spending time in subway systems seems to contribute to the personal airborne exposure to several metals. It has been reported that steel dust in the subway was the dominant source of airborne exposure to iron, magnesium, and chromium for many young people in New York (29) and spending time in the subway was a predictor for personal exposure to manganese in London (30) and Toronto (31). The toxicological importance of the quantities of iron in PM is dependent on the oxidation state. Analysis of crystalline compounds in our study indicated that the iron in the street particles was mainly in the form of hematite (Fe2O3) whereas iron from the subway particles was mainly magnetite (Fe3O4). This may explain the oxidative capacity of the subway particles since magnetite exhibits both Fe2+ and Fe3+ in a crystalline oxide structure. The mechanism behind the release of free radicals by particles most often discussed is the production of hydroxyl radicals in the presence of hydrogen peroxide and Fe2+ via the Haber-Weiss and Fenton reactions (32). There is likely an involvement of these kinds of reactions in our study since a large increase in oxidation was seen after the addition of H2O2 (Figure 2c). Furthermore, radical release from magnetite but not from hematite has been shown (32). The ROS formation by the iron-rich particles may also lead to increased particle uptake by the cells due to lipid peroxidation of cell membranes (33). Thus, high genotoxicity of the subway particles may be due partly to increased particle uptake. A close interaction of particles and lung cells in this study can be seen in the scanning electron microscopy images (Figure 4). In this study, we showed that the subway particles were approximately eight times more genotoxic and four times more likely to cause oxidative stress in cultured human lung cells. In addition, the particle concentration at locations where particles for this study were collected has been reported to be 5-10 times higher at the subway station as compared to the nearby very busy urban street (17). Thus, a relative in vitro comparison based on this study shows that the particles from one unit of air from the Stockholm subway may be in the range of 40-80 times more genotoxic and 20-40 times more potent to induce oxidative stress as compared to air from a busy urban street. However, the toxicity per unit particle and the intensity of exposure are not the only factors influencing the risk of disease for humans. The duration of exposure and the susceptibility of the exposed population are also important factors. Even though the exposure may be rather short in the underground, the intensity of the exposure and the high number of people using the underground (including sensitive people such as elderly and asthmatics), combined with the reactivity of the particles seen in this study, are good reasons for further examination of possible health effects caused by air particles in the subway system.

Acknowledgment. We thank Christer Johansson at the City of Stockholm Environment and Health Admin-

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istration for providing the particles. This study was financially supported by the Swedish Environmental Protection Agency and the Stockholm City Council. We have validated the method for analysis of 8-oxodG within ESCODD (European Standards Committee on Oxidative DNA Damage). The research group is a member of the European ECNIS (Environmental Cancer Risk, Nutrition and Individual Susceptibility) project.

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