Wear Particles Generated from Studded Tires and Pavement Induces

Particles from studded tire−pavement wear, generated using a road simulator, were able to induce inflammatory cytokines, NO, lipid peroxidation, and...
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Chem. Res. Toxicol. 2007, 20, 937-946

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Wear Particles Generated from Studded Tires and Pavement Induces Inflammatory Reactions in Mouse Macrophage Cells John Lindbom,† Mats Gustafsson,‡ Go¨ran Blomqvist,‡ Andreas Dahl,§ Anders Gudmundsson,§ Erik Swietlicki,| and Anders G. Ljungman*,† DiVision of Occupational and EnVironmental Medicine, Department of Molecular and Clinical Medicine, Faculty of Health Sciences, Linko¨ping UniVersity, SE-581 85 Linko¨ping, Sweden, Swedish National Road and Transport Research Institute (VTI), SE-581 95 Linko¨ping, Sweden, and DiVision of Ergonomics and Aerosol Technology and DiVision of Nuclear Physics, Lund UniVersity, P.O. Box 118, SE-221 00 Lund, Sweden ReceiVed January 15, 2007

Health risks associated with exposure to airborne particulate matter (PM) have been shown epidemiologically as well as experimentally, pointing to both respiratory and cardiovascular effects. These health risks are of increasing concern in society, and to protect public health, a clarification of the toxic properties of particles from different sources is of importance. Lately, wear particles generated from traffic have been recognized as a major contributing source to the overall particle load, especially in the Nordic countries where studded tires are used. The aim of this study was to further investigate and compare the ability to induce inflammatory mediators of different traffic-related wear particles collected from an urban street, a subway station, and studded tire-pavement wear. Inflammatory effects were measured as induction of nitric oxide (NO), IL-6, TNF-R, arachidonic acid (AA), and lipid peroxidation after exposure of the murine macrophage like cell line RAW 264.7. In addition, the redox potential of the particles was measured in a cell-free system. The results show that all particles tested induce IL-6, TNF-R, and NO, and those from the urban street were the most potent ones. In contrast, particles collected from a subway station were most potent to induce lipid peroxidation, AA release, and formation of ROS. Particles from studded tire-pavement wear, generated using a road simulator, were able to induce inflammatory cytokines, NO, lipid peroxidation, and ROS formation. Interestingly, particles generated from pavement containing granite as the main stone material were more potent than those generated from pavement containing quartzite as the main stone material. Introduction Several epidemiological studies have pointed to a connection between the levels of particulate matter (PM) in the environment, especially in urban areas, and respiratory and cardiovascular diseases (1-6). In addition, it was also recently shown (1) that particle exposure is associated with adverse effects on lung development in children. The cellular mechanism of these adverse health effects is unclear. Studies employing research animals and human cell lines, and in some cases autopsy results, have indicated an association between PM-induced oxidative stress and/or inflammation and health effects (7-11). Because of a lack of data regarding how particles from different sources contribute to effects on human health, particles are often handled as a uniform pollutant. Respirable particles are usually divided into three aerodynamic diameter categories: ultrafine ( > g > > > g

subway street subway subway subway street

> street ) ) subway ) > street > > street > > street > > subway )

subway subway subway subway subway subway granite street

g granite > g granite > ) granite > ) granite ) ) granite ) ) granite > > subway ) > granite ) granite granite granite granite granite granite

) ) ) ) ) )

quartzite quartzite quartzite quartzite quartzite quartzite quartzite quartzite quartzite quartzite quartzite quartzite quartzite quartzite

a Key: >, significant increase; g, trend that does not reach significance; ), no difference detected; and *, cell-free assay.

follows: CPM in medium/CPM in medium + CPM in cell solution × 100 ) % released AA. Analysis of Cell Viability. The viability of the cells was analyzed using the Trypan blue method. The cells were incubated for 15 min in the cell incubator with a 1:1 dilution of 0.1% trypan blue and PBS (PBS I), whereafter the cells were washed three times with PBS I to remove excess trypan blue before evaluation of the viability. Analysis of Endotoxin Content. The different PM types were analyzed for LPS content using Endotoxate (Sigma) according to instructions from the manufacturer. Statistical Analysis. A two-way analysis of variance test with Tukeys posthoc test was used when comparing the results for the cytokine, NO, lipid peroxidation, AA, and ROS induction. A Kruskal-Wallis test with Mann-Whitney posthoc test and Bonferroni correction for the mRNA analysis, p < 0.05, was considered statistically significant.

Results An overview of the results from the different inflammatory markers that were investigated is shown in Table 2. Secretion of Inflammatory Markers (TNF-r, IL-6, and NO). We have previously (18) noted that the particle extraction might result in the subsequent release of filter material and that this material can influence the cytokine release. Therefore, in a preliminary test, RAW 264.7 cells were exposed to granite, respectively, quartzite particles that had been recovered using shaking or water extraction, and the results were compared. The cells were exposed to 1, 10, and 100 µg/mL of the recovered material, and cytokine and NO levels in the culture medium were measured after 18 h of incubation. No difference between the two recovery methods was detected except for granite particles at the 100 µg/mL exposure level. At 100 µg/mL, granite shaken loose from the filter induced a 2.4 ( 0.3-fold higher (p < 0.0005) TNF-R secretion than what water-extracted granite did. Pieces of blank filters (new unused filters), respectively, filters with particles were cut out (4 cm × 4 cm) and treated with water extraction as described above and the amount of filter material in the particle samples was determined gravimetrically to 6 ( 1.7% of the weight. However, when cells were exposed to material recovered from blank filters, a significant effect on the NO and TNF-R induction was seen only at the 100 µg/mL exposure dose as compared to unexposed cells. Furthermore, when extracted material from visually particle free filters was used, only material from filters used to collect subway particles induced a significant release of TNF-R

as compared to unexposed cells and also first at the 100 µg/mL exposure level (742 ( 64 vs 224 ( 33 pg/mL, p ) 0.0005, mean ( SEM of three experiments performed as duplicates). No effect was noted on IL-6 secretion. Extracted material from subway filters, at the 100 µg/mL exposure level, was also able to induce a significant release of NO as compared to unexposed cells [p ) 0.004 vs control (unexposed cells)]. The values for the cytokines and NO when cells were stimulated with filter extracts from visually particle-free pieces of the filters used to collect the granite, quartzite, or street particles did not differ from the controls (data not shown). Because material originating from the filters only had effects on the TNF-R and NO release at the 100 µg/mL exposure level, it is unlikely that particles originating from the filter material significantly contribute to the effects seen after particle exposure. Furthermore, material originating from the filters had no effect on lipid peroxidation, ROS formation, or AA release. Therefore, in all subsequent experiments, unexposed RAW 264.7 cells were used as control and as comparison to the particle-exposed cells. All types of particles induced TNF-R secretion (Figure 2). Subway particles were the most potent ones inducing secretion already at 1 µg/mL (p < 0.0005) as compared to control (unexposed cells), while significance was reached at 10 µg/mL for granite and street (p ) 0.0005) and first at 100 µg/mL for quartzite (p ) 0.001). However, when comparing subway to street at the 1 µg/mL exposure level, no difference was detected. Respective particles ability to induce TNF-R secretion at the same dose is shown in Table 2. Street particles were the only ones inducing IL-6 at the 10 µg/mL exposure level as compared to control cells (Figure 3). Granite, quartzite, and subway particles were able to induce a significant (p < 0.05) IL-6 secretion first at the 100 µg/mL exposure level. Comparison between the particles potency for release of IL-6 at the same dose is shown in Table 2. Granite and street were the only particle types to induce significant (P < 0.0005) release of NO as compared to control (unexposed cells) (Figure 5a). Street released significantly (p < 0.0005) more NO as compared to granite for every dose of the same concentration (Figure 5b). Results of Inhibitors. DFX had a trend to inhibit TNF-Rinduced release by street and subway particles (Figure 2), which did not reach significance. However, an increasing effect for DFX in combination with quartzite was noted (Figure 2). Exposure of cells to DFX alone did not induce any TNF-R liberation. For all PM types, except for street particles, DFX significantly inhibited the induced IL-6 secretion (Figure 3). NAC significantly inhibited both TNF-R and IL-6 secretion for all of the PM types (Figure 2 and 3) and also suppressed the base levels of TNF-R below the detection limit of the assay when cells were exposed to NAC alone (p ) 0.004). No effect on the base levels for IL-6 could be observed for NAC. NO secretion from street particle-exposed cells (100 µg/mL) was inhibited to 61 ( 13% (p < 0.0005) by L-NAME, while the effect on the granite (100 µg/mL) induced NO secretion did not reach significance. Neither NAC nor DFX had any inhibitory effect on the NO secretion induced by granite or street particles, nor did they have any effect on the NO liberation from RAW 264.7 by themselves. Wortmannin in combination with granite or street increased the NO-inducing effect with 39 ( 18 (p ) 0.005) and 150 ( 24% (p ) 0.0005), respectively, but no effect on the nitrite production was seen when cells were exposed to wortmannin alone. Polymixin B did not inhibit NO secretion from cells exposed to granite or street particles or had any effect when incubated with the cells alone.

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Figure 2. Secretion of TNF-R from RAW 264.7 cells into the cell growth medium after exposure to different concentrations (µg/mL) of respective particle types alone or in combination with drug as described in the Materials and Methods. Each bar represents the mean ( SEM of three independent experiments performed as duplicates. Note differences in scale between the different PM types. * ) p < 0.05 vs control. # ) p < 0.05 for drug vs PM 100 µg/mL. β-Glucan (100 µg/mL) was used as the positive control inducing the release of 34643 ( 3120 pg/mL TNF-R. A significant (p < 0.05) increase in response was noted when comparing the 10 and 100 µg/mL doses for granite, quartzite, and street, while no dose response was noted for subway particles.

Figure 3. Secretion of IL-6 from RAW 264.7 cells into the cell growth medium after exposure to different concentrations (µg/mL) of respective particle types alone or in combination with drug as described in the Materials and Methods. Each bar represents the mean ( SEM of three independent experiments performed as duplicates. Note differences in scale between the different PM types. * ) p < 0.05 vs control. # ) p < 0.05 for drug vs PM 100 µg/mL. β-Glucan (100 µg/mL) was used as the positive control inducing the release of 3721 ( 543 pg/mL of IL-6. A significant (p < 0.05) increase in response was noted when comparing the 10 and 100 µg/mL doses, respectively, for all particle types.

Gene Expression. The effect of the PM types on the gene expression is shown in Table 3 and Figure 4. The mRNA of TNF-R showed a trend to increase for street particles but did not reach significance. The IL-6 gene expression increased for

granite (p ) 0.011), street (p ) 0.001), and subway (p ) 0.009) as compared to control but not for quartzite. Street was the only particle type able to induce (p < 0.05 vs control) iNOS mRNA (Table 3 and Figure 4).

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Figure 4. Gene expression in RAW 264.7 cells after 18 h of exposure to 100 µg/mL of respective particles. RT-PCR was performed as described in the Materials and Methods. Representative of three experiments performed as duplicates.

Figure 6. Induction of lipid peroxidation expressed as µM TBARS. One million cells were incubated for 18 h with 100 µg/mL of respective particles with unexposed cells as controls; p < 0.05 for all particle types. Each bar represents the mean ( SEM of at least three independent experiments performed as duplicates. No induction of TBARS as compared to control was induced by 100 µg/mL β-glucan. Iron sulfate, 20 and 200 µM, was used as the positive control, resulting in 0.32 ( 0.07 and 2.14 ( 0.18 µM TBARS, respectively. Analysis was performed as described in the Material and Methods.

Figure 7. Formation of ROS induced in a cell-free system by the different particle types. Values are expressed as % (mean ( SEM of three independent experiments performed as duplicates) of remaining absorbance. Analysis was performed as described in the Materials and Methods. Representative of three experiments performed as duplicates. The values for granite and quartzite were 87 ( 2.2 and 81 ( 1.0%, respectively, at the exposure dose 100 µg/mL, which is significantly lower as compared to the control. Figure 5. (a) Secretion of NO from RAW 264.7 cells after exposure to 100 µg/mL of respective particle types for 18 h shown as multiples of controls (unexposed cells) due to variation of NO secretion from control. * ) Significance (p < 0.05) as compared to control (unexposed macrophages). Each bar represents the mean ( SEM of at least three independent experiments performed as duplicates. Analysis performed as described in the Materials and Methods. β-Glucan (100 µg/mL) was used as the positive control inducing the release of 22.6 ( 3 more NO than unexposed cells. (b) Secretion of NO from RAW 264.7 cells after exposure to different concentrations of granite and street for 18 h shown as multiples of controls (unexposed cells) due to variation of NO secretion from control. * ) Significance (p < 0.05) as compared to control. # ) Significance (p < 0.05) as compared to the closest lower dose. Each bar represents the mean ( SEM of at least three independent experiments performed as duplicates. Analysis was performed as described in the Materials and Methods.

AA Release. Subway particles were the only particle type able to induce a detectable AA liberation from the RAW 264.7 cells, and this was seen only after exposure to 100 and 250 µg/mL (p ) 0.002, p < 0.0005 vs control, respectively). Illustrated in Figure 8, the AA liberation induced by subway particles was within the same magnitude as corresponding doses of β-glucan.

Lipid Peroxidation. All particle types induced lipid peroxidation measured as TBARS (Figure 6) as compared to unexposed control cells. Comparing the particle types showed that street and subway particles induced significantly (p ) 0.005) higher lipid peroxidation as compared to granite or quartzite particles (Table 2). One possible source of error is that the reaction takes place during the acidic incubation at 90 °C, when the cells have been lysed and iron ions released from the particles may initiate the formation of TBARS, despite the antioxidative effect of BHT. To investigate this possibility, PM was sonicated directly in TCA and added to RAW 264.7 cells lysed through sonication and incubated in 90 °C with TBA reagent. No effect of the particles was noted, which indicates that the lipid peroxidation was formed during the incubation time of 18 h. Incubation of the cells with DFX did not effect the induction of lipid peroxidation of the particles but impaired the effect of iron sulfate. ROS Formation (Redox Potential). All particle types induced ROS formation, although the ability varied strongly depending on particle type (Figure 7). While both street and subway particles induced a significant (p < 0.05 vs control)

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Table 3. Gene Expression of IL-6, TNF-r, and iNOS in RAW 264.7 Cells after 18 h of Exposure to Respective Particle Typesa particle type (µg/mL)

IL-6

TNF-R

iNOS

granite 10 granite 50 granite 100 quartzite 10 quartzite 50 quartzite 100 street 10 street 50 street 100 subway 10 subway 50 subway 100 β-glucan 10 β-glucan 50 β-glucan 100

1.0 ( 0.4 2.1 ( 0.4* 2.4 ( 0.9* 1.7 ( 1.1 1.7 ( 1.1 0.6 ( 0.2 1.8 ( 0.3* 3.9 ( 0.3* 4.5 ( 0.7* 0.4 ( 0.2 1.0 ( 0.2 0.8 ( 0.3 2.4 ( 0.2* 8.2 ( 2.6* 6.9 ( 2.1*

1.0 ( 0.4 1.4 ( 0.3 1.2 ( 0.5 3.6 ( 1.5 2.4 ( 1.3 0.7 ( 0.1 0.7 ( 0.2 2.0 ( 0.7 2.0 ( 0.4 0.7 ( 0.1 1.1 ( 0.1 0.7 ( 0.1 1.1 ( 0.1 4.4 ( 1.5 3.0 ( 1.2

0.4 ( 0.3 0.6 ( 0.3 0.6 ( 0.3 0.4 ( 0.2 0.5 ( 0.2 0.3 ( 0.1 0.3 ( 0.1 2.0 ( 0.7* 1.8 ( 0.5* 0.2 ( 0.1 0.4 ( 0.2 0.3 ( 0.1 0.9 ( 0.2 3.8 ( 0.4* 3.7 ( 1.0*

a All values are means ( SEM of five separate experiments. The amount of mRNA for each respective protein has been normalized against the 18s RNA amount in the same sample. The values for controls (unexposed cells) were as follows: IL-6, 0.2 ( 0.2; TNF-R, 1.1 ( 0.4; and iNOS, 0.2 ( 0.1. β-Glucan was used as a positive control. The analysis was performed as described in the Materials and Methods. * ) p < 0.05 vs control.

Figure 8. AA liberation, from RAW 264.7 cells, after exposure to subway particles (100 and 250 µg/mL, respectively) for 18 h. β-Glucan at equal doses was used as a positive control. The values (mean ( SEM of four independent experiments performed as duplicates) are expressed as % liberated AA of total AA incorporated. * ) p < 0.05 vs control. The other particle types did not induce any significant AA release over control. Analysis was performed as described in the Materials and Methods.

formation already at 10 µg/mL of each respective particle type, granite and quartzite induced a small but significant decrease in the absorption first at the 100 µg/mL exposure level. Both street and subway had a significant dose response for all concentrations tested. No difference between granite and quartzite was seen. When comparing the different particle types, there was a significant difference between the ability of the particles to induce oxidation of DTT. Subway particles were the most potent and reached significance (p < 0.005) over the other particles at all doses. Subway and street particles incubated with DFX prior to adding them to the cells had a lower ability to induce ROS formation as compared to particles without DFX. The ROS formation induced by street (50 µg/mL) and subway particles (10 and 50 µg/mL) was inhibited 17 ( 3 (p ) 0.018), 24 ( 0 (p < 0.05), and 31 ( 8% (p < 0.05), respectively, by DFX

(values are means ( SEM of three experiments performed as duplicates). Endotoxin Content. Only street particles showed a positive result for endotoxin content, which is in accordance with earlier investigations, where endotoxin has been associated with urban particles (31). According to the manufacturer, the E-TOXATE test should give positive results down to 0.05 EU/mL. To check for the presence of E-TOXATE inhibitors in the samples to be tested, a positive control of 0.2 EU/mL is added to each sample to check for E-TOXATE inhibitors. Our results indicate that granite, quartzite, and subway particles contain less than 0.05 EU/mL (∼0.01 pg/mL), while street particles contain 0.05 EU/ mL or more of endotoxin. None of the particle types contains E-TOXATE inhibitors. Cell Viability. The viability of the RAW 264.7 was not influenced by any of the particle types or concentrations used and were in all cases g90%, except for the combination of subway particles with NAC where the viability dropped to 20%.

Discussion The aim of this study was to investigate and compare different types of traffic-related wear particles regarding their inflammatory evoking potential and through the use of inhibitors gain some insight into the inflammatory mechanisms. The results show that all exposure to each PM sample used in our study resulted in the induction of inflammatory mediators, although there were variations among their potency to do so and that this ability also varied depending on the inflammatory marker measured. Even though exposure to particles is recognized as a health risk and several health effects have been observed in epidemiological studies, it is not clear which specific types of particles, size fraction(s), and concentration are the cause behind these observations, nor is the cellular mechanisms fully understood. Because urban PM consists of a cocktail of particles with different origins, it is in the interest of public heath, an important task in particle toxicology, to provide information that may clarify the toxic properties of particles from different sources. A previous study (17) in our laboratory using human macrophages showed that wear particles from studded tires and pavement can induce the release of proinflammatory cytokines within or above the same magnitude as particles collected at a traffic intensive street or at a subway station and that the stone material used in the pavement is of importance for this effect. In collaboration with other investigators, we have also, in a recent publication (18), showed that particles from road wear are genotoxic. However, they are far less genotoxic than subway particles. To further elucidate the influence of biological end point measured on the estimated relative toxicity, murine macrophage cells (RAW 264.7) were exposed to different concentrations of wear particles generated at the interface of studded tires and pavement, collected at a traffic intensive street or at a subway station, and examined for changes in cytokine, NO, and AA release. Furthermore, the cell viability, endotoxin content redox potential, and lipid peroxidation ability were examined. The results (summarized in Table 2) show the importance of measuring several biological markers indicating that the relative toxicological potential is dependent on the marker measured. A problem when performing this type of toxicological study is to avoid involvement of fibers released from the filters during the particle extraction. This study showed that glass filter fibers are able to induce TNF-R and NO secretion but have no effect on lipid peroxidation, ROS formation, or AA release from RAW

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264.7 cells. However, the amount of filter material constitutes 6 ( 1.7% of the weight, and a detectable effect was first seen when 100 µg/mL of “pure” filter material extracted from a blank filter was used. To further control for effects originating from filter material, filter extracts from filter pieces visibly judged to be free from particles were tested, and no effect was detected until the dose used was 100 µg/mL and this only when material extracted from a filter used to collect subway particles was used. Furthermore, when comparing the effects induced by particles that had been shaken loose from the filters to those that had been water extracted, no difference was seen. Thus, it is unlikely that fibers originating from the filter material contributed substantially to the effects seen. All particle types induced cytokine release with subway particles being the most potent, inducing TNF-R at the 1 µg/ mL exposure level, while street particles were the most potent when IL-6 was measured (Figures 2 and 3). For both cytokines, the highest dose (100 µg/mL) of granite induced a release of 2-3 magnitudes higher than quartzite, while at lower doses this effect was not so prominent (Figures 2 and 3). At the gene expression level, none of the particle types was able to significantly increase the amount of TNF-R or IL-6 mRNA (Table 3). It is possible that in this case the gene expression precedes the protein induction but also that the exponential phase of the mRNA level does not correspond to that of the protein. The ability of granite to induce a higher cytokine response at the same dose as quartzite from RAW 264.7 cells is a similar observation as for human monocyte-derived macrophages (17). This indicates that some of the chemical or physical properties of granite have a higher inflammatory potential than those of quartzite. However, the result showing that street is remarkably more potent at inducing TNF-R secretion from RAW 264.7 cells as compared to human monocyte-derived macrophages (17) is probably explained by a difference in sensibility between the two cell types. Street particles did not have an effect on RAW 264.7 cell viability, while monocyte-derived macrophage viability was reduced already at the 10 µg/mL exposure dose (17). It is also possible that endotoxin is responsible for at least some of the cytokine-inducing effects even though street particles were the only particle type containing detectable endotoxin. The inhibitor NAC was able to abolish the cytokine release, indicating oxidative stress as well as the transcription factors AP-1 and NF-κB in the underlying mechanism. NAC is known as a scavenger of ROS and to block several kinases resulting in inhibition of transcription factors such as AP-1 and NF-κB but may also exercise its inhibitory functions through other mechanisms (32, 33). However, the ability of NAC as an inhibitor of gene activation is not undisputable, as this reducing agent under certain conditions seems to be able to act as a NFκB activator (34, 35). DFX has been reported to act both as intracellular metal chelator and as a radical scavenger (36-38) and to inhibit TNF-R secretion (39, 40). However, our results show only a slight inhibitory effect on TNF-R induced by street or subway particles, and surprisingly, the combination of quartzite and DFX increased the TNF-R secretion (Figure 2). As for NAC, the effect of DFX seems to be divided, as it also has been reported to increase the production of TNF-R and other cytokines from peripheral blood mononuclear cells from cerebral infarction patients and in human mast cells by NF-κB and hypoxiainducible factor 1R activation (41, 42). This activation may be mediated by removal of the central iron of the putative heme oxygen sensor by DFX (41), and it is possible that certain material(s) in quartzite may facilitate this process. As DFX had

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no inhibiting effect on granite or quartzite, the TNF-R increase is probably not mediated by the small iron content or radical hydroxyl generation by these two types of PM, while it is possible that this is the case concerning street and subway particles. However, DFX significantly impaired the induction of IL-6 for all types of PM (Figure 3), and both the IL-6 and the TNF-R genes are activated through NF-κB. This may be due to the large difference in the concentration levels between the two cytokines, where a small effect of DFX is more prominent on the IL-6 formation or by different ways of activation of these two cytokines in this cell type, as there exist diverse combinations of the NF-kB subunits that are under the control of different types of NF-kB inhibitors. The result of the inhibitors does not point out any exact way of cytokine induction by the PM, as both of the inhibitors may exercise their effect in several ways. NAC completely abolished the base levels of IL-6 and TNF-R in cells exposed to NAC only, while DFX had no significant effect. This difference is probably due to the ability of NAC to block NF-κB. Only granite and street induced NO production. As the same types of tires were used when generating the wear particles, differences in the mineral composition between granite and quartzite are probably ascribed to this observation. Neither NAC nor DFX had any impairing effect on the induction of NO. Phosphatidylinositol 3-kinase (PI3K), which may activate endothelial NOS (42), is inhibited by wortmannin that has previously been reported to have an NO-inducing effect on RAW 264.7 cells in combination with LPS (43). This effect was also noticed in combination with granite or street, but no effect on the nitrite production was seen when cells were exposed to wortmannin alone. As street particles were shown to contain LPS by the endotoxate test, it is possible that the endotoxin content was responsible, but according to our results, granite did not contain any LPS. Also, polymyxin B did not reduce the NO secretion induced by street. This indicates that the NO release induced by granite and street may work through the same pathway as LPS, by NF-κB activation. Wortmannin had no inhibitory effect on NO secretion, and while this was observed for L-NAME, as well as the elevated iNOS, mRNA expression for street (Figure 4) suggests the activation of iNOS by the particles is the mechanism resulting in NO release. All particle types induced lipid peroxidation (Figure 6), and this toxic marker showed that the induction was within the same magnitude for both granite and quartzite. Subway particles induced the highest response, which could be expected due to the high iron content, but as DFX had no impairing effect on the lipid peroxidation, it is possible that other particle components are involved. One such component that is present in all particle types and that have been shown to induce lipid peroxidation is silica. Airborne particles have been shown to induce lipid peroxidation in previous investigations (19-47), and PM2.5 was able to evoke this toxic effect. It could therefore be expected that differences in the size distribution between the PM types also might be responsible for variation in the induced lipid peroxidation (17). As for lipid peroxidation, subway was also the dominant particle type in the cell-free ROS formation assay, which could be expected as ROS can induce lipid peroxidation (48). This is also in accordance with our previous results that showed subway particles to be the most genotoxic (18). The small (not statistically significant) effect of DFX on ROS formation induced by street and subway particles is most likely ascribed to the radical scavenging effect, rather than iron chelating as

Wear Particles From Tires and PaVement

the presence of hydrogen peroxide is necessary for Fenton or Haber-Weiss reactions. NAC was not used in the ROS experiments as the thiol group interferes with both DTT and DTNB. Only exposure to subway particles resulted in AA release, which was within the same order of magnitude as for β-glucan that was used as a positive control (Figure 8). Subway particles have recently been shown to pose a health risk by their ability to cause DNA fragmentation and oxidative stress (18, 49), and here, we show that they also have the ability to induce AA liberation. This result is in line with a previous study (21) showing AA release from RAW 264.7 cells after exposure to airborne city PM at similar doses (53 µg/cm2) as the doses used in this study. Because the viability of the cells after exposure to all particle types used in this study was equivalent, it is not likely that the AA release could originate from dead cells. Iron may increase the uptake of Ca2+ (50), and ultrafine particles may, independent of iron, increase intracellular Ca2+ (51). Thus, one possible explanation is that subway particles may activate one or more of the cytosolic PLA2 types through influx of calcium ions resulting in cleavage and release of AA (52). Unexpectedly small particles (around 10 nm) were generated during the trials with the road simulator (for details, see ref 28). Overall, these particles seem to have an organic origin, indicating a source other than the coarser particles. The mass of these particles constitutes only about 1%, but numerically, they are in the majority, and it cannot be ruled out that they contribute to the effects seen in this study. The authors are well aware of the fact that we do not know if the nanoparticles are present in the cell exposure experiments. Urban street particles and subway particles are known health risks, and our results indicate that PM generated from studded tire/pavement might be a contributing factor to the toxicity of urban street particles, by increasing the amount of PM generated as well as by mediating inflammatory effects. Notably, as shown in Table 2, street particles are more potent at inducing the release of cytokines and NO as compared to subway particles. However, subway particles are more potent at inducing lipid peroxidation, ROS formation, and AA release. This may partly be due to the endotoxin content of street, respectively, metal content in subway particles. Because the street particles were collected during early spring at a time point when studded tires were still in use and, therefore, may contain wear particles of the same type as those generated in the road simulator, this investigation also points to the importance of the pavement material in mediating toxicological effects of street particles. As with all cell-based models, the actual human exposure scenario in vivo is not possible to duplicate, and caution should be taken when human effects are considered. Nevertheless, because human exposure data, regarding toxic properties of particles from different sources, are lacking and our results were generated using exposure levels (10-100 µg/cm2) commonly used in a cell-based exposure system (21, 53), they might be a valuable contribution to the information considered when deciding how to cost effectively lower the health risks that the general population is exposed to by particles in the ambient air. Acknowledgment. We thank Thomas Lingefeldt, Linko¨ping University, for work with SEM, Christer Johansson for providing street and subway particles, and Tomas Halldin for invaluable operation and maintenance of the VTI road simulator. This work was supported by the Swedish Road Administration, Sweden.

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