Airway contraction and cytokine release in isolated rat lungs induced by wear particles from the road and tire interface and road vehicle brakes

Abstract The dominant road traffic particle sources are wear particles from the road and tire interface, and from vehicle brake pads. The aim of this work was to investigate the effect of road and brake wear particles on pulmonary function and biomarkers in isolated perfused rat lungs. Particles were sampled from the studded tire wear of three road pavements containing different rock materials in a road simulator; and from the wear of two brake pad materials using a pin-on-disk machine. Isolated rat lungs inhaled the coarse and fine fractions of the sampled particles resulting in an estimated total particle lung dose of 50 μg. The tidal volume (TV) was measured during the particle exposure and the following 50 min. Perfusate and BALF were analyzed for the cytokines TNF, CXCL1 and CCL3. The TV of lungs exposed to rock materials was significantly reduced after 25 min of exposure compared to the controls, for quartzite already after 4 min. The particles of the heavy-duty brake pads had no effect on the TV. Brake particles resulted in a significant elevation of CXCL1 in the perfusate. Brake particles showed significant elevations of all three measured cytokines, and quartzite showed a significant elevation of TNF in BALF. The study shows that the toxic effect on lungs exposed to airborne particles can be investigated using measurements of tidal volume. Furthermore, the study shows that the choice of rock material in road pavements has the potential to affect the toxicity of road wear PM10.


Introduction
Airborne particulate matter is a major health problem that is estimated to cause 4.14 million premature deaths worldwide each year (Fuller et al. 2022).The correlation between adverse health effects and particle concentrations in ambient air is well known, and recent studies have indeed found an association between particulate matter (PM) exposure and, for example, impaired lung function (Chen et al. 2019;Takebayashi et al. 2021, Fussell et al. 2022).Particle concentrations are often particularly high in urban areas, resulting in high exposure levels for many people.In these environments, emissions from road traffic are a major source to particulate matter (Pant and Harrison 2013).Several studies have described the importance of the effects of ambient traffic-related PM fractions on respiratory lung function and asthma (O'Connor et al. 2008;Guarnieri and Balmes 2014;Hwang et al. 2015;Chen et al. 2018).
The concentration of PM in the air at a given location is dependent on contributions from long-range transport as well as local sources.Recently, the non-exhaust particles from road traffic (i.e.particles generated from the wear of tires and road pavement), referred to as tire and road wear particles (TRWP) and those of vehicle brakes, have been recognized to be at least as high as exhaust emissions (Piscitello et al. 2021).In the Nordic countries in particular, where studded tires are used in the winter, the emission of road wear particles is the strongest source to PM10 in roadside environments during winter and spring (Hussein et al. 2008;Gustafsson et al. 2009;Kupiainen et al. 2016).Regulations are in force for particle exhaust pollution, which has been successively diminishing.There has also been an increase in the electrification of vehicles.At the same time, unregulated non-exhaust particle emissions are growing with the increase in traffic and due to trends, such as the use of heavier vehicles (SUVs and electric cars) and more powerful engines (Timmers and Achten 2016;Sommer et al. 2018;Baensch-Baltruschat et al. 2020;Beddows and Harrison 2021).Consequently, there is increasing concern about the health effects of non-exhaust particles from road traffic and the underlying mechanisms by which they exert their toxic actions.
The toxicity of PM particles is dependent on several factors such as chemical composition, shape, adsorbed materials, and particle sizes where PM fractions are usually divided into three particle size modes: ultrafine (�0.1 mm in diameter), fine (PM2.5-0.1, particles between 0.1 and 2.5 mm) and coarse (PM10-2.5, particles between 2.5 and 10 mm).TRWP are particles formed in, and emitted from, the tire and pavement interface and constitute a mix of particles originating in the pavement and tires but also from dust accumulated on the pavement.The particles generated from pavement wear mainly consist of minerals from the pavement rock material (R€ ais€ anen et al. 2003).As such, the types of minerals are important in determining the inflammatory potential of the road wear particles, where in-vitro cell studies of mylonite, for example, have shown it to be more potent than quartz, basalt and feldspar (Schwarze et al. 2002).The particles generated from granite road pavement had higher inflammatory potential than those from quartzite road pavement (Lindbom et al. 2006).Coarse particles (PM10) from the studded tire wear of pavements have induced several pro-inflammatory events in murine RAW 264.7 macrophages (Lindbom et al. 2006).Øvrevik et al. (2005) performed a cell in-vitro study of nine different rock materials.It showed that some of the particle fractions, such as fine (<2.5 lm) and coarse (<10 lm) (sieve fractions), induced the release of high levels of macrophage inflammatory protein (MIP)-2 from T2 cells, and high levels of IL-8 from small airway epithelial cells (SAECs).There were no consistent differences related to size fractions, indicating that other factors are more important for inflammatory potential.However, no particular mineral or element was related to the chemokine release.Mice exposed through the inhalation of re-aerosolized PM2.5 particles from brake pads and wear particles from studded tires and pavement (generated during the same campaign used in the present study) showed no lung cytotoxicity or systemic oxidative stress.In the bronchoalveolar lavage fluid (BALF), though, there was an induction of pro-inflammatory responses for certain brake pads, but not for wear particles from studded tires and pavement (Gerlofs-Nijland et al. 2019).Different tires may also produce different effects as previously shown, where passenger tire particles induced higher genotoxicity and inflammatory responses in a RAW 264.7 cell line compared to truck tire particles (Poma et al. 2019).
Another aspect to keep in mind is that wear processes can also generate ultrafine particles related to heat generation.Ultrafine brake wear particle emissions are associated with a threshold temperature (Jansson et al. 2010a;Nosko and Olofsson 2017;Vojt� ı� sek-Lom et al. 2021) and tire wear results in ultrafine particle emissions at harsh braking, acceleration, or steering (Mathissen et al. 2011;Kwak et al. 2014), or when using studded tires (Dahl et al. 2006).
Ultrafine brake wear particle emissions contain a volatile fraction (Perricone et al. 2019) and can also be generated by pure thermal effects without any friction (Ma et al.2020).Ultrafine particles may cause pulmonary toxicity after inhalation.This was recently shown for some engineered nanoparticles where increases of neutrophil chemo-attractants were suggested to be potential biomarkers of pulmonary toxicity following particle exposure (Tomonaga et al. 2020).
Studies of tidal volume may be useful to gain more knowledge about the underlying mechanisms in the lungs when dealing with particle exposure.Tidal volume is the amount of air that moves in or out of the lungs with each respiratory cycle.It is a vital clinical parameter that allows for proper ventilation to take place (Hallett et al. 2021).Chuang et al. (2020) studied the associations between soluble metals and lung and liver toxicity in mice induced by fine particulate matter.This showed that PM2.5 significantly decreased the tidal volume after exposure.Another important effect of particle exposure is pro-inflammatory responses (R€ onkk€ o et al. 2021).
The aim of the study presented in this paper was to investigate and improve our knowledge about the biological and toxicological effects of wear particles generated from road pavements containing the different rock materials used in Swedish road pavements, and from road vehicle brake materials.This was done in terms of how the elemental composition and particle size biomarkers affected the pulmonary functions and the release of biomarkers of isolated and perfused rat lungs.

Generation, sampling and characterization of tire and road wear particles
The tire and road wear particles from studded tire wear of road pavement were generated, sampled and characterized as previously described (Gustafsson et al. 2008).Briefly, a road simulator (see Supplement S1) was used to generate the tire and road wear particles from the tire/pavement interface.Studded Nokian Hakkapeliitta 7 tires were used to wear the pavements at 70 km/h.Stone mastic asphalt (SMA) pavements with a maximum aggregate size of 11 mm were used for the tests.This construction is common on highly trafficked Nordic roads because it is very wear resistant and can resist rutting for a long time.The rock aggregates were chosen to have noticeably different mineralogy so that we could correlate any differences in toxic results with the rock composition.The rock aggregates were also chosen to test relevant alternatives that could be used on Swedish roads.The rocks used were diabase, quartzite and syenite from Swedish quarries.All of them fulfilled the above-mentioned criteria.
The generated airborne particles were sampled for particle characterization by using a PM10 inlet (Ruppert and Patashnick, U.S.A.).They were then divided into three flows with an isokinetic splitter and measured using an Aerodynamic Particle Sizer (APS model 3021, TSI Inc., U.S.A.), and a 13-stage cascade impactor for succeeding size-resolved element analyses using PIXE (Particle Induced X-ray Emission) with a GeoPIXE spectrum fitting (Shariff et al. 2002).Elements heavier than aluminum could be analyzed.Measured particle size distributions and element composition is shown in Supplement S2.
Particles were sampled for toxicological tests using a highvolume cascade impactor (HVCI, Harvard School of Public Health) supplied by RIVM (Rijksinstituut voor Volksgezondheid en Milieu, The Netherlands).The HVCI sampled two fractions: coarse (PM 10-2.5 ) and fine (PM 2.5-0.1 ) in polyurethane foams (PUF).The sampled particles were extracted using a standard protocol (Gerlofs-Nijland et al. 2005).Particles were stored in airtight vessels (no inert gas was used) at −80 � C in the dark until the exposure experiments.

Generation, sampling and characterization of road vehicle brake wear particles
The generation of brake particles was performed using a pinon-disk machine (see Supplement S1) at KTH Royal Institute of Technology, equipped with a horizontal rotating disk and a dead-weight-loaded pin (load 110 N, rotating speed 3 000 rpm, mean contact pressure 0.28 MPa) mounted in a sealed box to sample air that only contained wear particles (Wahlstr€ om et al. 2010).Two types of brake pads were used.One from a light duty vehicle including a low metallic brake pad and one from a heavy-duty vehicle.Both pads were worn against a disk from a cast iron rotor.The light duty disk brake was representative of a typical medium-size European passenger car and the heavy-duty disk brake assembly was representative of a typical European long distance truck.The pin samples of 10 mm in diameter were machined from real brake pads.Equally, disk samples of 63 mm in diameter, and 6 mm thick, were machined from a real disk braking surface, The brake particle size distributions were measured and sampled with the same equipment used for the TRWP (tire and road wear particles) and analyzed using PIXE.

Experimental particle exposure design
The lungs from rats were excised and treated in ten different groups: nine groups were exposed to rock particles (diabase, syenite, quartzite) and to light duty (LD) and heavy duty (HD) brake particles.The remaining one was an unexposed control group (see Table 1).All the exposed groups were divided into fine and coarse particles.A minimum of five rat lungs in each exposed group were successfully perfused and ventilated, except for the LD brake fine particles (only 2 rat lungs) and the HD brake fine particles (none).There were 46 experiments in total.

Particle aerosol generation and lung exposure
A Vilnius Aerosol Generator (VAG, CH Technologies Inc., U.S.A.) was used to disperse the particles (see Figure 1).The output particle concentration was then controlled by a self-designed LabVIEW program.It used the measured particle concentration in the exposure chamber with a DustTrak photometer (model 8520, TSI Inc., U.S.A.) to produce a constant particle concentration and final intended dose.An aerosol concentrator (a virtual impactor) concentrated the aerosol into 2 Lpm, which passed a humidifier (RH between 50% and 80%) and a neutralizer (Kr-85, 74 Figure 1.The experimental set-up for redispersion of the sampled rock and brake particles and delivery to the exposed rat lung via an exposure chamber. Table 1.The different lung exposed groups and the number of which were exposed to fine and coarse particles.MBq, TSI Inc., U.S.A.) before entering the exposure chamber of 0.15 liter.An APS (see above) was used to measure the particle size distribution.The DustTrak measured the particle concentration in the exposure chamber and was calibrated against gravimetric filter sampling.There was a weak over-pressure in the exposure chamber that generated a small excess aerosol flow of 0.5 Lpm.
The rat lung inhaled the aerosol from the exposure chamber through a straight stainless-steel tube, which was injected into the trachea tube of the rat lung (see details below).A desirable total lung dose was 50 lg corresponding to 0.2 mg/kg body weight.To reach the target, Equation 1was used to calculate the necessary inhaled particle mass concentration (c).The Multiple Path Particle Dosimetry (MPPD) model was used to calculate the particle deposition efficiency of the rat lung (DEP eff ).The MPPD used a mass median diameter of 2.1 lm and 4.2 lm values for the fine and coarse particle fractions, respectively, along with a geometric standard deviation of 1.7 and a tidal volume of 2.0 ml, resulting in a DEP eff in the rat lung of 24% for fine particles and 40% for coarse particles, respectively.The values of the different parameters were based on corresponding measured mean values.The breathing frequency (f breath ) was regulated to a fixed value of 72 per minute.
The total deposited rat lung dose (D) was calculated using the equation: Where c � (mg/m 3 ) is the measured inhaled mean particle mass concentration (the concentration in the exposure chamber), TV (cm 3 ) is the measured mean tidal volume, and T exp (s) is the exposure time summarized for each partial exposure (i) of the total number of n.The tidal volume measurements are described in the section "Measurements of rat lung airflow and tidal volume."

Chemicals
Bovine serum albumin (fraction V) was used from the Sigma Chemical Company, St. Louis, MI, U.S.A.All chemicals were of analytical grade.

Animals
Male Sprague-Dawley rats aged 5-6 weeks (170 grams) were obtained from Taconic Europe, Denmark, and used for all studies.The animals were derived from a pathogen-free colony and housed under pathogen-free conditions in the Animal Care Center of Link€ oping University.They were allowed to acclimate for 4-6 days under a 12/12-h light/dark cycle in isolated cages (4-6 rats in every cage) with filtered clean air ventilation before the experiments.All animal procedures were approved by the Animal Care Panel of Link€ oping University.

Isolated perfused and ventilated rat lung (IPRL) preparation
Rats were anesthetized with an intraperitoneal injection of pentobarbital sodium (50 mg/kg).The lungs were ventilated and perfused through the heart and were excised (Lindahl et al. 1991;Uhlig and Wollin et al. 1994;Baker et al. 1999).The lungs were suspended by the trachea in a humidified, artificial thoracic chamber at 37 � C and ventilated by negative pressure ventilation with 72 breaths per minute −1 (Nosratabadi et al. 2003), see Supplement S3.The lungs were perfused at constant hydrostatic pressure (10 cm H 2 O) through the pulmonary artery with a Krebs-Ringer buffer (in mM; NaCl 118.0, KCl 4.7, NaHCO 3 24.9,KH 2 PO 4 1.2, CaCl 2 2.5, MgSO 4 1.2) containing 12.5 mM Hepes, 5.0 mM glucose, and 2% bovine serum albumin fraction V.The lungs were perfused with a total volume of 50 mL of recirculating buffer.Left arterial pressure (pulmonary outflow) was set to 0 cm H 2 O by adjusting the height of the outflow reservoir.The thoracic chamber pressure was measured with a differential pressure transducer (Validyne DP45-14) and the airflow velocity was measured with a Pneumotachograph tube (Fleisch type 0000 with heater, 230 vac) connected to a differential pressure transducer .A deep breath was initiated every 5 min by increasing the end-inspiratory pressure to −15 cm H 2 O.The differential pressure transducers were connected to differential pressure amplifiers (Gould Instrument System) and the data were transmitted to a computer and collected using the Data Sciences International PNM-P3Plus software.

Measurements of rat lung airflow and tidal volume
After preparation, all lungs were perfused and ventilated for 15 min, during which they stabilized.Lungs that showed no signs of unstable pulmonary inspiratory airflow during the stabilization period were used for further experimentation.The data from the pneumotachograph tube airflow velocity were entered into the PNM-P3Plus software to calculate tidal volume (TV) at any given time (TV t ).A normalized tidal volume was calculated as TV t /TV 0, where TV 0 is the tidal volume recorded after the 15 min of stabilization and before the particle exposure was performed.

Procedure of rat lung particle exposure
Once the lungs were stabilized (15 min) and their functionality was assessed to be normal, the pneumotachograph tube was replaced with the exposure chamber, and the rat lungs started to breathe the particles they were exposed to. Figure 2 shows a typical result from a pneumotachograph measurement.After 4 min of exposure, the exposure chamber was removed and the pneumotachograph was reconnected.The airflow was then measured for 30 s, and the lung took a deep breath.The exposure chamber was then reconnected to the lungs, and they were exposed to particles for another 4 min.The airflow was measured again.This procedure was repeated 5 times.It resulted in TV t at the following time points: 5, 10, 15, 20 and 25 min.This means that during a running time of 25 min, the effective exposure time was 20 min.After the particle exposure, the lungs breathed through the pneumotachograph for another 50 min in which a deep breath was initiated every 5 min.The control group lungs were prepared in the same way as those in the particle study groups, but without any particle exposure.The pneumotachograph and the exposure chamber were connected to the lungs and the tidal volume was monitored during the entire study period.For the analysis of tidal volume, a mean of 12 breaths 30 s before a deep breath was initiated and was calculated throughout the measurements.
For the fine particle exposed group, the pneumotachograph airflow measurements during particle exposure were limited in order to reduce the running time due to reduced access to fine particles materials.In some experiments, the dispersion of fine particles was not adequately sufficient, and the effective exposure time had to be prolonged from 20 min up to 30 min.

Measurements of cytokines
To examine the effects of inhaled particles on the release of inflammatory cytokines, perfusate samples were taken at the beginning of the experiment before exposure, immediately after exposure, and at the end of the experiment.BALF was also obtained from particle-exposed groups and the control group at the end of the experiment.Directly after perfusate sampling and lung lavage, the samples were frozen at −82 � C until the day of cytokine analysis.
To investigate the eventual effect of the particle exposure on inflammatory markers, a screening of the potential markers was carried out in the perfusate and BALF from the quartzite-exposed rat lungs using the Proteome Profiler Rat Cytokine Array (Panel A, R&D Systems, Minneapolis, MN, U.S.A.).The screening followed the manufacturer's instructions, with the exception that the resulting chemiluminescence was detected directly using a VersaDoc 4000MP system (Bio-Rad Laboratories, Hercules, CA, USA).Three cytokines showed a qualitative-determined presence in the BALF and a time-dependent increase in the perfusate (see Supplement S4).They were selected for subsequent analysis and quantification across all samples using a commercially available enzyme-linked immunosorbent assay (ELISA) kit.
The cytokines tumor necrosis factor (TNF), growth-regulated alpha protein (CXCL1, also called cytokine-induced neutrophil chemoattractant-1, CINC1), and the C-C motif chemokine 3 (CCL3, also called macrophage inflammatory protein 1 alpha, MIP1a) were measured in BALF and all perfusate samples by sandwich ELISAs, as supplied in the kits, according to the manufacturer's instructions (R&D Systems, Minneapolis, MN, U.S.A.).Samples were analyzed in duplicate.Limit of detection (LOD), defined as average þ2SD of 16 replicate blank samples optical density, on each of the assays was as follows: TNF (1.7 pg/ml), CXCL1 (3.6 pg/ml) and CCL3 (0.7 pg/ml).

Statistical analysis
All toxicological data are expressed as the mean ± standard error of the mean (SEM).The toxicological responses regarding the tidal volume and cytokine analyses were expressed as the ratio between the value at each measured time point and the initially measured value before exposure (time 0), that is, individually normalized values.Linear correlations between the normalized tidal volumes and cytokine analysis were tested.A general linear repeated measures model was used to analyze the differences in changes of outcomes between the different exposures for the tidal volume measurements.Additionally, linear correlation analysis was performed to test the relationship between tidal volume and the two variables body weight and individual dose.Analyses were performed using SPSS software (Statistics version 26, IBM, U.S.A.).For the cytokine analysis, the Mann-Whitney U-test was used to compare each exposed group vs. the control group in Statistica (v.13.5.0.17,Tibco Software).The significance level was set at 0.05.Typical diagram of the air velocity measurement using a pneumotachograph connected to a rat lung.After 15 min of stabilization, the lung was exposed to particles, typically for 25 min.This was followed by 50 min of post-exposure during which a deep breath was initiated every 5 min.During coarse particle exposure, the pneumotachograph was connected to the rat lung 4 times for short 30 s measurements.The vertical gray lines show each of the 5-min occasions when the tidal volume was measured during 12 deep breaths (10 s).

Airborne particle characterization
The normalized particle mass distributions of the quartzite pavement PM10 sampled in the exposure chamber, and the mass distribution when the quartzite pavement PM10 was collected at the road simulator using a high volume sampler with a PM10 inlet are presented in Figure 3A and corresponding data for LD brake particles are shown in Figure 3B.Table 2 presents the estimated mass median diameter (MMD) and geometric standard deviation (GSD) for all nine exposed groups as well as estimated doses.The size distributions were measured using the APS and all the diameters were aerodynamic particle diameters, using a density of 2.8 g cm −3 for the density correction of the APS.The estimated deposited rat lung doses (D) shown in  Table 2 were calculated using Equation 1.The mean value of the deposited lung doses for all the fine and coarse particle exposures were 63 lg and 39 lg, respectively.The relative standard deviations of the mean values for the different groups of fine and coarse were 22% and 27%, respectively.Most of the variation was due to variation in the tidal volumes between the rat lungs.
Figure 4 shows the ratio of coarse to fine fractions for quartzite and LD brake particles in the exposure chamber.This was measured using the APS.The particles were originally collected using a heating, ventilation, and airconditioning (HVAC) system designed for the separation of fine particles (0.1 to 2.5 lm) and coarse particles (2.5 to 10 lm). Figure 4 shows that the exposure experimental setup used in this study succeeded in regenerating and reproducing these size fractions for the exposure of isolated and perfused rat lungs.
For all the material used, both the fine and coarse modes were analyzed using PIXE, which analyze elements higher than aluminum.Figure 5 shows the relative concentrations of the most abundant elements in the fine fractions for each of the three rocks and the two brake materials.The rock materials consisted mostly of Si (47-60%), S (0.1-10%), K (3-20%), Ca (5-21%), Fe (9-24%) and small fractions of Cr  (0.01-0.2%),Mn (0.1-0.3%), Ti (0.8-1.9%), Cu (0.08-0.24%) and Zn (0.2%).The largest differences (not shown) between fine and coarse modes for all rock materials were higher contents of Cu in the fine mode: diabase 3.0 times higher, quartzite 5.9 times higher, and syenite 4.5 times higher than in the coarse mode.There were also larger concentrations of S and Cl.The fine fractions for quartzite had a higher content of Cr and Ni (4.8 and 14.42, respectively) compared to the coarse fractions.The LD and HD brakes consisted mostly of Fe (64 and 71%) and Cu (8 and 4%) and small fractions of Si (9 and 1%), S (2 and 5%), Ca (2 and 1%), Ti (0.1 and 3%), Cr (0.1 and 2%), and Zn (0.2 and 2%).Note that graphite is commonly used in the brake pad formulations and can be up to 10 wt%.The friction material components, often more than 20%, are embedded in a phenolic resin that also contains carbon.A current important change regarding brake pads for passenger cars is related to recent regulations aiming at reducing the use of copper down to 0.5% by 2025 (Lyu et al. 2020).

Studied animals
All the rats in each of the 10 groups studied were from the same batch and were used mostly during one week after they were delivered to the Animal Care Center of Link€ oping University.From each batch, we used the lungs of two or three of the rats as unexposed control lungs.Usually, two rat lungs were used in every experiment day.We used a total of 85 rat lungs, of which 69 (46 exposed and 23 controls) were studied for 75 min, and 16 were excluded.Fifteen lungs were excluded after the 15 min stabilization period because they did not meet the functionally normal lung criteria.One lung was excluded because of technical problems.The control group experiments were spread over the whole study period to minimize differences between different batches of animals.The mean body weights for all nine exposed groups were between 218 and 257 grams and the mean value for the control group was 287 grams.The body weight of all 69 rats varied from 202 to 417 grams.The average body weight and number of rats studied in each group are presented in Supplement S5.The results of the statistical analyses of the relationship between body weight and tidal volume showed no significance for either the exposed or control groups.This can be due to the constant under-pressure in the chamber.Consequently, the body weight was not included as a covariate in the following analyses performed with the general linear model.

Effect of particle exposure on tidal volume
The effects of the particle depositions of coarse quartzite, syenite, diabase, LD and HD brakes on the test groups' tidal volumes were compared to the unexposed control group.The results are shown in Figure 6.The tidal volume of the control group is unchanged for the first 35 min., but at 40 min., it significantly decreases (p ¼ 0.016).After that, there is a linear decrease in the tidal volume.After 45 min., all three rock materials show a slope similar to that of the controls.No significant differences in the slopes were observed.
The coarse quartzite particles already had a statistically significant decreasing effect on tidal volume in the isolated lungs after the first dose of particle exposure, which means a 4 min.exposure corresponding to 20% of the total dose, versus the controls (p < 0.014).For subsequent times, the decrease of the tidal volume is statistically significant with p-values less than 0.005.For syenite, a significant (p ¼ 0.04) decrease of tidal volume compared to the controls can be seen at time 20 min., corresponding to 80% of the total dose.For diabase, a significant difference (p ¼ 0.02) can be seen first after 25 min., corresponding to 100% of the total dose.From 35 to 75 min.,there were significant differences (p < 0.005) at each time point between all coarse rock particle groups versus the controls.
The statistical analysis of the tidal volume measured between 35 and 75 min.shows no significant differences between the coarse particles of all three rocks at any time, except for quartzite and syenite at 35 and 75 min.(p ¼ 0.042 and p ¼ 0.041, respectively).The heavy duty (HD) and the light duty (LD) brake particles had no effect on tidal volume during the first 45 min.After that, the tidal volume of the LD brake particles changed more dramatically (bigger slope) compared to all the rock particles.After 65 min., the LD brake particles had a tidal volume comparable to the rock particles (Figure 6).HD brake particles showed no effect on tidal volume at any time compared to the control particles.
The effects of fine and coarse particle exposures on the lungs are shown as normalized tidal volumes in Figures 7A,  7B, 7C and 7d.
For all the three rock materials there are no statistical differences between fine and coarse particles at any time after 35 min (Figure 7A, 7B and 7C).However, there were significant differences (p < 0.005) at each time point from 35 to 75 min.between all the fine rock particle groups versus the controls, except for syenite at 55 to 75 min.Before 35 min.,only measurements of tidal volume were performed for fine particles for diabase and syenite.For the diabase, though, differences were observed between fine and coarse particles.At 10 and 20 min., there was a trend of decreasing values for fine diabase compared to coarse diabase, but it was not statistically significant (p ¼ 0.20 and p ¼ 0.10, respectively).
The effect on tidal volume from fine particle exposure to LD brake material (no fine particle exposure to HD brake material was performed) is similar to that of the fine rock materials, namely a significant effect after 35 min., despite the effect on tidal volume of coarse LD brake particle exposure that was delayed until 60 min.However, only two exposures to fine particles from LD brake material were performed, which is why conclusions should be drawn with caution.Based on the limited range of fine and coarse particle doses, no effect by them could be observed on tidal volume.

Effects of particle exposure on pro-inflammatory cytokines
The exposure of lungs to LD brake material particles resulted in a significant elevation of CXCL1 in the perfusate of the exposed rat lungs compared to the unexposed controls at all the measured time points (Figure 8B).Diabase and HD brake particle exposure caused a single significant increase of CXCL1 at the 25 min.time point while the levels were similar to the controls at t ¼ 75 min (Figure 8B).LD brake particles also showed higher CCL3 at timepoints 0 and 25 compared to the unexposed controls (Figure 8C).TNF showed no significant differences between the exposed groups and the controls (Figures 8A).
Regarding BALF collected at the end of the experiment, LD brake particles showed significant elevations of CXCL1 and CCL3, while HD brake particles showed a significant elevation of TNF compared to the controls (Figure 9A-C).Quartzite showed a significant elevation of TNF as compared to the controls (Figure 9A).Normalized tidal volume (TV) for different rocks and brake particles for fine and coarse particles.The TV of a given time point was normalized to the TV at time zero (TV t /TV 0 ).error bars present the ± standard error of the mean.The ventilated rat lungs were exposed to the particles after 15 min. of stabilization.The particle exposure started at time zero.For all coarse particles, the exposure time was 25 min.For fine particles, the exposure time varied between 20 and 35 min.due to technical issues.For the fine particles of diabase and syenite, the number of TV measurements until 35 min.were reduced compared to the number given in the legends, but never less than 3.

Relationship between tidal volume and cytokine analysis
Analyses were performed for 25 min.and 75 min.,corresponding to the time points when the rat lung perfusates were collected.Generally, low correlations (r 2 <0.5) were observed between normalized tidal volumes and normalized cytokine levels (TNF, CXCL1 and CCL3) for all rock and brake materials at both 25 and 75 min.However, for coarse brake HD and CXCL1 at 75 min.,there was a negative correlation (r 2 ¼ 0.84): higher CXL1 concentrations were correlated to lower tidal volumes.No correlations were found at 25 min.(r 2 ¼ 0.01).

Usability with IPRL
To investigate the biological effects of tire and road wear particles and brake pad wear particles, cell based or whole animal methods have been used in different studies (Kreider et al. 2012;Gerlofs-Nijland et al. 2019).In the present study, we chose to use an isolated perfused rat lung (IPRL) model to study the effects on pulmonary functions of different wear particles that had been re-aerosolized under controlled conditions in a laboratory environment.Unlike studies that are in-vitro cell-based, the IPRL is an ex-vivo model that offers unique possibilities to study the effects of different agents on the respiratory and pulmonary immune system and on different types of immunocompetent cells in their native environment.Compared to whole animal studies, the pulmonary structure and functional integrity remains intact without the influence of immune cells and cytokines in blood and other organs.Moreover, unlike the intratracheal instillation in animal studies, the interaction between the airborne particles and the lung cells/tissues, takes place during realistic breathing conditions.The method also offers the ability to sample perfusate that circulates through the lungs to detect and analyze cytokines that have passed from the alveoli to blood vessels during the experiment period.Finally, lung lavage sampling can be carried out after the study time period to analyze changes in different cytokines.To our knowledge, the IPRL technique has mostly been used to investigate the pulmonary pharmacokinetics of inhaled therapeutics (Mobley and Hochhaus, 2001;Sakagami 2006;Fernandes and Vanbever 2009).
In the present study, we demonstrated that the IPRL technique together with particle re-aerosolizing and the exposure model that we designed, can be used to study the health effects of tire and road wear particles that are generated and collected in different fractions in a controlled environment and re-aerosolized during the IPRL exposure.By extending the exposure time over a longer period (25 min) and performing frequent measurements of tidal volume (calculated from air velocity measurements using pneumotachograph), we have shown that dose-response relationship can be measured.The used methodology involves methanol extraction, sonication and evaporation of sampled particles, which should be considered when comparing the results to other studies where other extraction and sonication methodologies have been used.

Effect on tidal volume and influence of different rock and brake materials
The effect on tidal volume can be described as two processes.One occurred for all the lungs, including the controls, and was expressed as a linearly decreasing tidal volume after 60 min.for all the lungs studied, which depends on the experimental set-up itself.The lungs were stressed by gravity and lost their elasticity, which obstructed the ability of the lungs to expand when the under-pressure in the chamber increased.The second process occurred when there was a sudden decrease of the tidal volume compared to that of the control group.This happened for all the particle exposures that were studied (except for the light duty brake particles), but at different time points and for different particle doses that entered the rat lungs.
The quartzite coarse particles had a significant effect on pulmonary airways by decreasing the tidal volume just after the lungs were exposed for 20% of the total exposure dose, compared to syenite and diabase that showed decreased tidal volumes after 80% and 100%, respectively.
The increasing airway contraction and decreasing tidal volume can be due to the rock particles' toxicity and irritative capacity by releasing different cytokines (Øvrevik et al. 2005).This may be the oxidative effect caused by the inhalation of respirable particles in the pulmonary airways (Knaapen Ad, 2004).Oxidative stress has been put forward as a central hypothetical mechanism in the adverse effects of PM, including ultrafine particles (Donaldson et al. 2003).Schaumann et al. (2004) studied the oxidative capacity of PM on the bronchial inflammatory response after instillation in normal human volunteers.It is generally accepted that the toxicity of quartz is caused by the reactive particle surface.Previous studies have shown that the bioactivity of quartz particles of different origins may vary substantially (Donaldson et al. 2003).An interesting observation was the much bigger slope of the tidal volume for coarse LD brake particles at 60 min., and that the fine fractions had similar effects on rock materials at 35 min.
Tidal volume measurements during fine particle exposure (time 0 to 25 min, which sometimes was extended to 35 min due to technical issues) were performed to a limited extent and only for diabase and syenite.For fine diabase particles, a trend of lower tidal volume was observed compared to the coarse particles.This decrease is similar to the fast response to coarse quartzite particles on tidal volume.However, for tidal volumes measured after 35 min no statistical differences were observed between exposure of fine and coarse rock particles.

Effect on biomarkers and influence of different rock and brake materials
Cytokine measurements indicated that the brake particles, especially LD, were more potent in inducing CXCL1 in lung perfusate as well as BALF compared to the rock particle exposures.CXCL1 is part of the CXC family of chemokines and is known to be involved in the recruitment and activation of neutrophils.Its expression has previously been shown to increase in rats exposed to PM2.5 particles (Shi et al. 2019).Our increase of CXCL1 in perfusate as a result of break particles, but not rock materials, exposure can be explained by the fact that the break particles mainly consist of metals (such as iron, copper, tin and antimony) that are well known initiators of inflammatory processes in the lung (Lui et al. 2019).Furthermore, a recent study showed that dust generated from brake pads causes oxidation and inflammation in macrophages (Selley et al. 2020).Increased levels of CXCL1 and CXCL2 have been found in the BALF from mice 24h after being exposed to brake wear particles, although only in particles generated from brake pads that have less Fe but higher levels of Cu and other metals such as Ti, Zr, Ba -compared to more traditional brake pads (Gerlofs-Nijland et al. 2019), such as those used in our study.The difference in Gerlofs-Nijland's results compared to ours may be related to: 1) their use of different species, 2) that their exposure time was for 1.5-6h; and 3) that their markers were measured 24h post exposure.The increase of CXCL1 that was identified in our study supports the notion that the assessment of cytokine-induced neutrophil chemo-attractants as biomarkers may be useful for predicting pulmonary toxicity after particle exposure, as previously suggested for nanoparticles (Tomonaga et al. 2020).
Among the exposures to rock particles, quartzite showed a significant TNF increase and a non-significant trend for increases in both CXCL1 and CCL3 in the lavage fluid.This indicated the possibility of different effects in the lungs compared to diabase and syenite.In a previous study, using particles generated in the road simulator, it was shown that human derived macrophages and airway epithelial cells exposed to different particles (including quartzite) secrete different cytokines including TNF-a.Interestingly in that study, the quartzite particles seemed to be relatively less able to induce a cytokine response when compared to granite or street particles (Lindbom et al. 2006).Gerlofs-Nijland et al. (2019) used PM2.5 samples from the same quartzite pavement sampling campaign as were used in the current study.They found that tire/road wear had a low inflammatory response (neutrophil concentration, CXCL1 and CXCL2 in mice BALF 24h post exposure) compared to the low-metallic brake wear particles and no obvious dose response.These differences compared to our study may be explained by the methodological differences described above.

Relationship between tidal volume and biomarkers
Pro-inflammatory responses are considered important mechanisms in the lung as a result of particle exposure (R€ onkk€ o et al. 2021).Moreover, it has been shown that PM2.5 significantly decreases the tidal volume (Chuang et al. 2020).Together, this implies that there could be a link between tidal volume and cytokine expression.In the current study, we only detected a correlation in the small subset of coarse HD brake particles (n ¼ 5) and tidal volume at one time (t ¼ 75 min).As such, it appears that the eventual link between tidal volume and lung cytokines are at best weak or altogether independent.Interestingly, a new study exploring the adverse outcome pathway (AOP) of inhaled substances discusses that when particles reach the alveoli, the lung surfactant function is inhibited leading to alveolar collapse, reduced tidal volume, and thereby decreased lung function (Da Silva et al. 2021).This could be a possible mechanism by which the particles in the current study reduced the tidal volume.
The results indicate that both choice of rock materials in road pavements and brake pad materials can influence on the toxicity of generated inhalable particles, and can be considered a health-related factor to consider in road and street environments where many people are exposed to high road wear particle concentrations, such as in high-trafficked streets in cities.
The mean body weight particle concentrations for fine and coarse groups were 0.25 mg/kg and 0.16 mg/kg, respectively.Compared to other studies, e.g.Gerlofs-Nijland et al. (2019) exposure of mice to similar brake particles, our exposures are in the range of theirs, corresponding to very low 0.1 mg/kg or low 0.35 mg/kg exposure (their high dose was 1.6 mg/kg).Fedan et al. (2020) performed instillation rat studies of sand dust and MIN-U-SIL with a low dose at 0.8 mg/ kg and a high dose at 2.5 mg/kg.This means that our observed effects have been found at a dose that corresponds to the lower levels of other studies.It should be underlined that the exposure, if transferred into human exposure, correlates to several months of exposure to ambient air in urban environments, however the aim of the study was to compare toxicity between the tested particles, not compare to human health effects.

Implications for choice of rock and brake materials
In Sweden and other countries where pavement wear is a major contributor to high particle concentrations in traffic environments, the choice of road pavement is crucial for urban air quality and for reaching the environmental quality standards for air quality set up in the EU.Even though the specific toxicity of wear particles is not regulated, choosing both low-emitting and low-toxic materials should be prioritized from a health perspective.According to the results in this study, all rock materials have negative effects, but from a health point of view, syenite or diabase should be chosen before quartzite.
Brake wear is, on a global scale, an important particle source in traffic environments and work is ongoing both to reduce (Perricone et al. 2018) and regulate emissions.As road wear, the concentrations are highest in cities with high traffic amounts and a high frequency of braking (Harrison et al. 2021).The potential toxicity of the generated brake wear particles has been addressed by phasing out copper content until 2025, but the current toxicity of the very diverse composition of brake pads is not well known.

Conclusions
The overall results of this study show that the choice of rock material in road pavements as well as the type of brake pads used on road vehicles, has the potential to affect the toxicity of road traffic related PM10 and should be considered in environments where concentrations and exposures are high.From this study the following conclusions were made: � Quartzite particles had a larger effect on tidal volume, which was shown by its fast response and at a lower dose compared to all other studied particles (statistically significant lower tidal volume at 5-minute exposure representing approximately 20% of total doses).� The brake particles, LD to a higher degree than HD, induced more effects on the investigated biomarkers compared to the rock particles.� Based on tidal volume data after 50 minutes, there were no statistically significant differences between fine and coarse particles in all exposed groups.� We have demonstrated that the toxic effect to lungs exposed to different kinds of airborne particles can be studied by the combination of delivery of the airborne particle over a 25-minute period (not a single, short and high dose) and repeated measurements of tidal volume.

Figure 2 .
Figure 2. Typical diagram of the air velocity measurement using a pneumotachograph connected to a rat lung.After 15 min of stabilization, the lung was exposed to particles, typically for 25 min.This was followed by 50 min of post-exposure during which a deep breath was initiated every 5 min.During coarse particle exposure, the pneumotachograph was connected to the rat lung 4 times for short 30 s measurements.The vertical gray lines show each of the 5-min occasions when the tidal volume was measured during 12 deep breaths (10 s).

Figure 3 .
Figure3.(A) Normalized particle mass distributions for fine and coarse particle modes in the exposure chamber during quartzite exposure and corresponding mass distribution when collecting quartzite pavement PM10 at the road simulator.(B) Normalized particle mass distributions for fine and coarse particle modes in the exposure chamber during LD brake exposure and corresponding mass distribution when collecting LD-brake PM10 particles at the pin-on-disk machine.

Figure 4 .
Figure 4.The ratio of coarse to fine quartzite and LD brake particles during exposure.

Figure 5 .
Figure 5.The most abundant elements in the fine fractions of the three rock materials and two types of brake particles (light duty and heavy duty).Others include e.g.P, Sn and Sb.

Figure 6 .
Figure6.Normalized tidal volume (TV) for coarse particle exposure over time of three rock and two brake materials, and a control group.The TV of certain time points was normalized to the TV at time 0 (TV t /TV 0 ).error bars present the ± standard error of the mean.The ventilated rat lungs were exposed to the particles after 15 min of stabilization.The particle exposure started at time zero and continued for 25 min.

Figure 7 .
Figure7.Normalized tidal volume (TV) for different rocks and brake particles for fine and coarse particles.The TV of a given time point was normalized to the TV at time zero (TV t /TV 0 ).error bars present the ± standard error of the mean.The ventilated rat lungs were exposed to the particles after 15 min. of stabilization.The particle exposure started at time zero.For all coarse particles, the exposure time was 25 min.For fine particles, the exposure time varied between 20 and 35 min.due to technical issues.For the fine particles of diabase and syenite, the number of TV measurements until 35 min.were reduced compared to the number given in the legends, but never less than 3.

Table 2 .
Mass median diameter, geometric standard deviation (GSD) and total particles dose, fine and coarse, respectively, for the different exposed groups.