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# A toxicological comparison of tire and road wear particles emitted on rural and urban roads, alone or with the co-exposed pollutant benzo[a]pyrene: Size and origin as key factors in controlling the in vitro proinflammatory response

Abderrahmane Bouredji, Laurence Lumière, Riadh Lakhmi, Bogdan Muresan-Paslaru, Jérémie Pourchez, Valérie Forest

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Abderrahmane Bouredji, Laurence Lumière, Riadh Lakhmi, Bogdan Muresan-Paslaru, Jérémie Pourchez, et al.. A toxicological comparison of tire and road wear particles emitted on rural and urban roads, alone or with the co-exposed pollutant benzo[a]pyrene: Size and origin as key factors in controlling the in vitro proinflammatory response. Environment International, 2025, pp.109920. 10.1016/j.envint.2025.109920. hal-05359304

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# A toxicological comparison of tire and road wear particles emitted on rural and urban roads, alone or with the co-exposed pollutant benzo[a]pyrene: Size and origin as key factors in controlling the *in vitro* proinflammatory response

Abderrahmane Bouredji<sup>a, b</sup>, Laurence Lumière<sup>b</sup>, Riadh Lakhmi<sup>c</sup>, Bogdan Muresan-Paslaru<sup>b, d</sup>, Jérémie Pourchez<sup>a</sup>, Valérie Forest<sup>a, *</sup>

## ARTICLE INFO

Handling Editor: Adrian Covaci

### Keywords:

- Non-exhaust emissions

- Tire and Road Wear Particles (TRWP)

- Direct and combined cell toxicity

- Proinflammatory response

- Road surface contaminants

## ABSTRACT

Tire and road wear particles (TRWP) make up a significant and increasing proportion of non-exhaust emissions and contribute to air pollution. This study examines how the size and origin of TRWP affect their toxicological profile. For this purpose, macrophages were incubated for 24 h with fine (0.39–0.69 µm) or coarse (4.2–10.2 µm) TRWP collected on-road either in urban or rural areas. Incubations were carried out in the presence or absence of benzo[a]pyrene (B[a]P, an archetypal road traffic gaseous pollutant) to investigate possible combined toxic effects. The *in vitro* cell response was evaluated in terms of cytotoxicity (LDH release), proinflammatory response (TNF-α production) and oxidative stress (reactive oxygen species (ROS) production). No cytotoxicity and no oxidative stress were induced by TRWP, whatever their size and origin. However, all types of TRWP (urban or rural and in both sizes) were capable of triggering a dose-dependent proinflammatory response. A similar toxicity profile was observed with or without B[a]P. For fine emissions, urban TRWP elicited more proinflammatory effects than rural TRWP, while for coarse emissions, rural TRWP were more proinflammatory at low dose. These results, coupled with the study of the TRWP's chemical composition, reveal that: i) both the size and the origin of TRWP are key factors in their toxicity, and ii) the origin-driven toxicity stems from at least two sources. One source is the abrasion of tires and road materials, and the other is the particulate road traffic emissions, ambient pollution and road surface contaminants incorporated into TRWP.

## 1. Introduction

Air pollution is a major public health problem, with excessive concentrations of ozone and fine particles blamed for > 4 million premature deaths worldwide every year (European Environment Agency, 2023; Miller et al., 2012; Miller and Newby, 2020; Son et al., 2020; WHO, 2014). In fact, correlations between exposure to particulate matter (PM) and adverse health effects such as respiratory and cardiovascular diseases and cancers have been widely reported (Cazzolla Gatti, 2021; Charloux et al., 1997; Hamra et al., 2014; Kurt et al., 2016; Mayoralas-Alises and Diaz-Lobato, 2012; Nishida and Yatera, 2022; Raaschou-Nielsen et al., 2013; Tseng et al., 2019; Wong, 2014).

Pollution from road traffic is a major contributor to air pollution, especially in urban areas (Evangeliou et al., 2020). PM from road traffic originate either from exhaust emissions (EE, from the tailpipe) or from non-exhaust emissions (NEE, resulting from mechanical abrasion of brakes and tires, wear of road surfaces, and resuspension of road dust into the atmosphere by passing traffic) (Fussell et al., 2022; Harrison et al., 2021; Lewis et al., 2019; Piscitello et al., 2021). Although NEE emissions are becoming increasingly prevalent in urban areas, and a fast growing body of literature on their emission dynamics and environmental fate is becoming available (ADEME, 2022; Amato et al., 2014; Baensch-Baltruschat et al., 2020; Fussell et al., 2022; Harrison et al., 2021; Lewis et al., 2019; Piscitello et al., 2021), very little data exists on

<footnote><sup>a</sup> Mines Saint-Etienne, Univ Jean Monnet, INSERM, U 1059 Sainbiose, Centre CIS, F-42023 Saint-Etienne, France</footnote>

<footnote><sup>b</sup> Université Gustave Eiffel, AME-EASE, F-44344 Bouguenais, France</footnote>

<footnote><sup>c</sup> Mines Saint-Etienne, Univ Lyon, CNRS, UMR 5307 LGF, Centre SPIN, Saint-Etienne F-42023, France</footnote>

<footnote>* Corresponding author at: 158 cours Fauriel, CS 62362, 42023 Saint-Etienne Cedex 2, France.
E-mail address: vforest@emse.fr (V. Forest).</footnote>
their potential human health effects (Bouredji et al., 2023; Forest and Pourchez, 2023).

Tire and road wear particles (TRWP) make up a non-negligible and highly variable proportion (between <5 % and >30 %) of NEE particles (OECD, 2020). They are generated by friction between the tire tread and the road surface, resulting in tire tread abrasion. They therefore consist of tire wear fragments embedded in a matrix of road surface compounds and include environmental pollutants (Harrison et al., 2021; Lewis et al., 2019). TRWP emission rate and composition depend on many parameters such as the chemical composition of tires, the type of road surface, the driving style, the climate, the vehicle type and its weight, the contaminants present on the road surface, etc. (Baensch-Baltruschat et al., 2020; Thorpe and Harrison, 2008; Zhang et al., 2023). The size of TRWP is also an important factor to consider as it varies from <0.1 µm, which allows them to penetrate the alveoli (Löndahl et al., 2006), to several tens or hundreds of micrometers.

In a previous study, we have shown that tire particles could elicit a proinflammatory response in a macrophage model without causing significant cytotoxicity or increased reactive oxygen species (ROS) production (Bouredji et al., 2024b). This effect depended on the size of the tire particles (Bouredji et al., 2025). Due to the population's widespread and chronic exposure to various pollutants, the *in vitro* toxicity of tire particles was further examined in the presence of exhaust gases and benzo[a]pyrene (B[a]P); a major representative of polycyclic aromatic hydrocarbons (PAH) (Begeman and Colucci, 1968). In fact, B[a]P has an increased affinity for surfaces and is hydrophobic (Log KOW of 6.1 at 25 °C) (NIH, 2025), making it conducive to secondary exhaust particles formation and adsorption on NEE, including TRWP. The obtained data demonstrated that, depending on the size of the tire particles, the combined proinflammatory effects of tire particles and B[a]P were either additive or synergistic (Bouredji et al., 2024a).

Since the origin of TRWP can also have a major impact on the *in vitro* effects they induce (playing simultaneously on the chemical composition of TRWP, the presence of contaminants, etc.), the aim of the present study was to assess the impact of the origin of TRWP (urban or rural) on their toxicological profile, in the presence (or not) of B[a]P.

## 2. Materials and methods

### 2.1. TRWP collection

TRWP were collected on the road using a fully instrumented Renault Clio 3 (1.6i 16 V, 111 hp) equipped with a high-volume air sampler (HVS, HiVol Plus from Tisch) (Fig. S1A). The vehicle was fitted with standard summer tires (Michelin™ Energy Saver, 185/60 R 15 88H). Sampling took place in the area of Nantes (western France). Two types of environments were crossed: urban and rural (Muresan et al., 2024). The trips took place either in the Loire Valley Region of western France or within the Nantes metropolitan area (47°12'15"N, 1°32'35"W) (Fig. S1B). To ensure representative data and a sufficient particulate collection, the distance traveled during each trip exceeded 100 km. All trips took place under comparable weather conditions: clear sky with light wind (wind speed less than 5 m/s) and no rain during the previous days. The air temperature and relative humidity ranged from 10 °C to 20 °C and from 40 % to 90 %, respectively. In addition to weather and traffic conditions, the apparent characteristics of the road surface (i.e., texture, construction material and/or dust contamination) were manually recorded by the co-pilot (De Oliveira et al., 2024; Muresan et al., 2024).

Briefly, the air intake for TRWP collection was placed behind the front wheels on the passenger side (De Oliveira et al., 2024). It was positioned and oriented according to Rajaratnam and Walker (Rajaratnam and Walker, 2019) and Noh et al. (Noh et al., 2021) to capture particles originating from both the tire-pavement interface and the resuspension caused by the tires. The HVS suction velocity at the sample inlet (3.5 m/s) was between the average airflow velocity at the

rear of the wheels in urban areas (about 1.6 m/s) and on motorways (up to 6.1 m/s). The HVS unit was equipped with a cascade impactor consisting of six stacked impaction plates with mean cut-off diameters ranging from 0.69 µm to >10 µm (specifically > 10.2, 4.2, 2.1, 1.3, 0.69 and 0.39 µm) for a nominal airflow rate of 566 L/min. A slotted cellulose collection substrate was located on each plate to collect the particles and prevent them from bouncing off on impact. Particles with a median diameter of <0.39 µm that made it through the cascade impactor were collected using a glass fiber terminal filter with an apparent pore size of approximately 0.1 µm. To assess a size effect of TRWP, this study compared the proinflammatory responses of the coarse and fine fractions collected on the 4.2–10.2 µm and 0.39–0.69 µm cellulose substrates, respectively. Depending on which environments were crossed, both fractions accounted for a variable, yet usually significant, proportion of TRWP's PM₁₀ emissions: ranging from 10 % to 30 % and from <1 % to 25 %, respectively (Muresan et al., 2024).

For the extraction of the particles, the exposed cellulose substrates were dried in a desiccator for 72 h and weighted (we extracted 4 substrates, one substrate per condition: rural 0.39–0.69 µm, rural 4.2–10.2 µm, urban 0.39–0.69 µm and urban 4.2–10.2 µm). They were then placed in 10 mL of ultrapure water for 30 min each under rotative agitation, followed by a sonication in an ultrasonic bath (30 min, 130 kHz, 35 °C). These steps were repeated twice. The substrates were removed, placed in a desiccator for 72 h and reweighted to determine the mass of TRWP recovered by subtraction (= mass of filter before extraction – mass of filter after extraction); this mass was then related to the extraction volume (10 mL) to obtain the TRWP concentration in this initial solution. Visually, the filters remained intact after extraction, suggesting that little material from the filter entered the solution and that our samples consisted mainly of particles. Before use for the toxicological assays, the solutions containing the TRWP were sonicated for 2 min using a sonicator (20 kHz, 400 W, 70 % amplitude, Digital Sonifier® Units, model S-450D, Branson Ultrasonics, USA), with the water bath cooled using ice.

### 2.2. Benzo[a]pyrene

We selected B[a]P as an prototype of PAH (Bukowska et al., 2022; Dussert et al., 2020). B[a]P is a ubiquitous environmental pollutant. It is most prevalent in urban and industrial areas, where it circulates as atmospheric particles produced by incomplete combustion of organic matter as well as coal or petroleum distillation (Mastral and Callén, 2000). Like other PAH, B[a]P is a carcinogen and potential mutagen that poses a health risk to urban populations (Dickhut et al., 2000; Larsen and Baker, 2003). Its high evaporation temperature and hydrophobicity translates in an increased affinity for solid and particulate organic substrates, favoring its trapping in aerosols and particulate emissions. Various mechanisms of action of B[a]P have been reported, including the formation of DNA adducts, the generation of ROS, the activation of the aromatic hydrocarbon receptor (AhR) and various epigenetic changes. B[a]P is initially metabolized by cytochrome P450 (CYP P450) to a carcinogenic metabolite: 7β, 8α-dihydroxy-9α, 10α-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE), which forms DNA adducts and causes mutations and malignant transformation (Bukowska et al., 2022). Furthermore, once activated by B[a]P, the AhR transcription factor migrates from the cytoplasm to the nucleus, where it can induce the transcription of numerous genes involved in immune regulation, detoxification and proliferation, leading, for example, to the formation of ROS and proinflammatory cytokines (Bukowska et al., 2022). Therefore, it is vital to investigate the combined toxicity of TRWP and B[a]P. The B[a]P working solution used in this study was purchased from Fisher Scientific (Illkirch, France). The conditions for cell exposure to B[a]P are described in detail in section 2.4.1.
## 2.3. Physicochemical characterization of the particles

Elemental chemical composition, particle size and morphology were determined by Hitachi SU5000 scanning electron microscope (SEM) observations, coupled with energy dispersive X-ray (EDX) analysis of the metals (Muresan et al., 2024).

Prior to analysis, each filter underwent a 4–5 nm thick platinum plating to limit charge build-up on the sample surface. This resulted in clearer, more coherent images. The improved signal-to-noise ratio resulting from metallization therefore led to better detection of secondary electrons. Each point was selected manually. All elemental analyses were performed with a primary electron beam of 15 kV and an X-ray accumulation time of 120 s per analysis point (or zone). It is important to note that the elemental composition of emissions obtained by micro-EDX at an analysis point (or zone) is only semi-quantitative.

## 2.4. Toxicological assays

### 2.4.1. Cell culture and exposure to TRWP and benzo[a]pyrene

The RAW 264.7 cell line was derived from mouse macrophages transformed by Abelson murine leukemia virus and was provided by the ATCC Cell Biology Collection (Promochem, LGC, Molsheim, France). Cells were cultured in Dulbecco Modified Eagle Medium (DMEM) supplemented with 10 % fetal calf serum and 1 % penicillin-streptomycin at 37 °C in an atmosphere humidified with 5 % carbon dioxide. The cells were seeded in 96-well plates (100,000 cells in 50 µL medium per well) and allowed to attach overnight. The TRWP were diluted in the cell culture medium to achieve the different final concentrations (9 – 113 µg/mL). It should be mentioned that it is very difficult to determine the TRWP concentration to which we are exposed and it is therefore very difficult to simulate relevant exposure doses. However, as our aim was not to assess the risk of TRWP, but to assess their hazard, we did not necessarily mimic physiological doses but used a wide range of concentrations to potentially show a dose-dependent response. This range is standard in toxicology studies and is consistent with previous recommendations (Drasler et al., 2017). After a 24 h incubation with the particles, the cell response was evaluated in terms of cytotoxicity (assessed by the release of lactate dehydrogenase), proinflammatory response (measurement of TNF-α production) and induction of oxidative stress (assessed by the production of reactive oxygen species, ROS).

To study the combined effects of pollutants we also incubated cells with TRWP in the presence of B[a]P. We chose a 20 µM concentration based on literature data (Gao et al., 2022; Ji et al., 2013; Lin et al., 2020). B[a]P was dissolved into cell culture supernatant, simultaneously with the addition of TRWP. After a 24 h incubation, the cell response was evaluated as previously mentioned.

We did not perform specific tests, such as the Limulus Amebocyte Lysate (LAL) assay, to confirm that our samples were free from microorganism contamination. However, we are confident that any potential contamination did not affect our experiments, as the particles were diluted in cell culture medium supplemented with antibiotics (1 % penicillin-streptomycin).

### 2.4.2. Cytotoxicity test

Cytotoxicity was assessed by analyzing cell membrane integrity by measuring the release of the cytoplasmic enzyme lactate dehydrogenase (LDH) in cell culture supernatant using the CytoTox-96™ Homogeneous Membrane Integrity Assay (Promega, Charbonnières-les-Bains, France) according to the manufacturer's instructions. The optical density of the samples was determined using a microplate reader set at 450 nm (Multiskan RC; Thermolabsystems, Helsinki, Finland). It is worth mentioning that TRWP can interfere with LDH released from the cells, leading to biased results. This is especially true since signal detection is based on absorbance (optical density) measurements. To avoid this optical distortion, we centrifuged the cell culture after 24 h of contact between cells and particles. We used only the cell supernatant to

measure optical density. This medium did not contain TRWP (which remained in the pellet), thus limiting TRWP/LDH interference. Three independent experiments were performed in quadruplicate, and the activity of the released LDH was compared with the activity of negative control cells (incubated without particles). The maximum cellular LDH released after lysis of the cells served as a positive control.

### 2.4.3. Oxidative stress

The OxiSelect™ ROS Assay Kit (Euromedex, Mundolsheim, France) can be used to assess a wide range of ROS activities. The assay utilizes the conversion of a non-fluorescent substrate, 2,7'-dichlorodihydrofluorescein diacetate, which can easily diffuse through cell membranes and be converted to a fluorogenic molecule 2,7'-dichlorodihydrofluorescein (DCF) in the presence of ROS, with the amount of fluorescence directly related to the ROS content. Fluorescence was measured using a Fluoroskan Ascent fluorometer (Ex: 480 nm, Em: 530 nm, Thermolabsystems) after a 90 min and a 24 h incubation of the cells with the particles. Incubation of the cells with H₂O₂ (1 mM) was used as a positive control. Three independent experiments were performed in quadruplicate, and ROS formation was compared with that of the negative control (cells incubated without particles).

### 2.4.4. Proinflammatory response

After 24 h of contact between particles and cells, the production of TNF-α, a proinflammatory cytokine, in cell culture medium was determined using a commercial ELISA kit (Quantikine® Mouse TNF-α Immunoassay; R&D Systems, Lille, France) according to the manufacturer's instructions. The optical density of each sample was determined using a microplate reader set at 450 nm (Multiskan RC; Thermolabsystems, Helsinki, Finland). As before, because TRWP can interact with cytokines, we centrifuged the cell culture after 24 h of contact between cells and particles, using only the cell supernatant to measure optical density. This medium did not contain TRWP, thus limiting TRWP/cytokine interaction. A standard curve was established and the results were expressed in picograms of TNF-α per milliliter of supernatant. Three independent experiments were performed in quadruplicate, and TNF-α production was compared with that of control cells (incubated without particles).

## 2.5. Statistical analysis

Analyses and graphs were performed using Prism software (GraphPad, San Diego, CA). Significance was established with two-way ANOVA tests and Tukey post-tests (p < 0.05). Each data point represents the mean of at least three independent experiments each performed in quadruplicate and is presented with the arithmetic standard error of the mean (± SEM).

# 3. Results

## 3.1. Physicochemical characterization

The morphology and chemical composition of the collected particles differ significantly, both between size fractions and between environments crossed. Microscopic observation and gravimetric analysis of exposed substrates indicate that TRWP emitted during the urban trip are more prevalent in the fine fraction (25 ± 2 % vs. 13 ± 1 % in the rural area). Conversely, the rural trip is characterized by an increase in supermicron TRWP, accounting for up to 80 % of emission mass locally (compared with <60 % in urban areas). Nevertheless, the mass-weighted median diameters of urban and rural emissions remain of a similar order: i.e., 3.4 ± 0.2 µm and 4.6 ± 0.3 µm, respectively. This indicates that changes in mass-weighted size distribution arise from a limited number of large particles, which are also found in urban areas, albeit less frequently and with less marked diameters (Fig. 1).

EDX analyses revealed that the chemical composition of TRWP
<figure><img src="image_3.png" /><figcaption>Fig. 1. Observation of urban or rural TRWP of 0.39–0.69 µm or 4.2–10.2 µm by scanning electron microscopy.</figcaption></figure>

depends on the particles' origin (Table 1) and size. In short, the coarse and fine TRWP fractions collected during the urban trip contained heavy metal-enriched particles, primarily titanium (Ti), copper (Cu), chromium (Cr), nickel (Ni), tin (Sn), zinc (Zn), and/or vanadium (V), in that order. This reflects the local abundance of anthropogenic sources, such as building materials, exhaust emissions from vehicles and industries, brake dust, and white road marking paint. On the one hand, the enrichment of urban TRWP emissions with metals commonly found in brake dust is more pronounced in the finest fraction. The ratio of metal content between the fine (0.39–0.69 µm) and coarse (4.2–10.2 µm) TRWP fractions ranges from 1.3 for iron (Fe) to greater than 5 for Cu and beyond 20 for Sn and Zn. On the other hand, coarse TRWP exhibited higher contents of Ti, Cr and Ni. While the presence of Ti is likely due to the wear of white road marking paint, Cr and Ni are thought to stem from a common industrial source (Wang et al., 2023). Elements enriched in TRWP from rural areas are Fe, Ti, Cr, silicon (Si), calcium (Ca), aluminum (Al), magnesium (Mg), manganese (Mn) and potassium (K). They mainly pertain to the local coarse TRWP fraction and account for contaminants from nearby soils (Fe, Si, Ca, Cr, Al or Mn, from x1.1 to x2.4), as well as road marking paint (Ti, factor of x1.2), oceanic fallout and winter road salting (Mg and K, from x1.9 to x4.6).

Another significant difference between TRWP of different origins and size fractions is that coarse urban TRWP have a higher content in tire fragments than rural TRWP (6.1 ± 0.9 % vs. 1.4 ± 0.2 %), while the opposite is true for the fine fraction (2.3 ± 0.3 % vs. 6.1 ± 0.8 %). On the one hand, this reflects the role of road surface contaminants as a third abrasive body in rural areas, which intensifies the abrasion of fine TRWP and mixes them with various dust deposits of terrigenous or oceanic origins. On the other hand, in urban areas, large rubber fragments torn from tire treads are generated during series of accelerations and braking, tight bends made at reduced speeds, and stop-and-go situations (Muresan et al., 2024). Lastly, is also worth mentioning the presence of

cerium (Ce), lanthanum (La) or niobium (Nb) embedded in both fine and coarse urban TRWP (Figs. S2). These elements may have been detached from the washcoat of diesel oxidation and three-way catalysts in vehicles.

## 3.2. Toxicity assessment

Initially, it was considered using a wide range of TRWP concentrations throughout toxicological assays to demonstrate possible dose-response effects as a function of TRWP origin and size fraction. However, limited and variable amounts of TRWP were obtained due to the differences in emission rates and yields of TRWP extraction from HVS collection substrata (extraction yields ranged from 14 % to >100 %). Consequently, data is missing in toxicity plots (particularly for rural trip) and TRWP concentrations achieved were reported with a standard deviation: i.e., 9 ± 1, 18 ± 3, 30 ± 4, 60 ± 7 and 113 ± 7 µg/mL corresponding to 5 ± 1, 11 ± 2, 18 ± 2, 36 ± 4 and 68 ± 4 µg/cm² respectively. Although some concentrations are missing, we always have the 4 experimental conditions: rural 0.39–0.69 µm, rural 4.2–10.2 µm, urban 0.39–0.69 µm and urban 4.2–10.2 µm to study the effects of the origin and size of TRWP on their toxicological profile.

### 3.2.1. Cytotoxicity

Fig. 2 shows the relative cytotoxicity of TRWP alone or in the presence of B[a]P as determined by the LDH release.

No significant difference in LDH release was observed between unexposed cells and the cells incubated with TRWP, whatever their size, origin and concentration. It suggests that TRWP are not cytotoxic. Similarly, cells incubated with B[a]P alone or in combination with TRWP did not shown any sign of cytotoxicity.

### 3.2.2. Oxidative stress

The Fig. 3 represents the relative ROS production induced after 90 min and 24 h respectively of contact between cells and TRWP. The ROS production was also assessed in the presence B[a]P.

After a 90 min incubation of cells with TRWP, no significant increase in ROS production was observed, suggesting that TRWP were not able to induce oxidative stress, irrespective of their origin, size, concentration or the presence of B[a]P. When incubation lasted 24 h, the ROS production was slightly higher but was not statistically different from that of control (unexposed) cells.

### 3.2.3. Proinflammatory response

#### 3.2.3.1. Impact of the benzo[a]pyrene. Fig. 4 reports the

<caption>**Table 1**</caption>

The main elements enriched in the urban or rural TRWP (qualitative analysis of chemical element composition by EDX).

<table><thead><tr><th></th><th>Urban TRWP</th><th>Rural TRWP</th></tr></thead><tbody><tr><td>Coarse TRWP</td><td>Ti (0.1–26 %), Cr (&lt; 0.1–6.3 %), Ni (0.4–3.7 %), Mg (&lt; 0.1–5.0 %), V (&lt; 0.1–0.2 %), Ce, La and Nb (inclusions)</td><td>Fe (0.7–60 %), Si (&lt; 0.1–13.9 %), Ca (0.2–9.6 %), Cr (1.0–6.3 %), Al (&lt; 0.1–6.7 %), Mn (0.6–0.8 %), K (&lt; 0.1–2.2 %)</td></tr><tr><td>Fine TRWP</td><td>Cu (0.2–7.9 %), Ni (0.4–1.3 %), S (&lt; 0.1–31 %), Ba (0.6–42 %), Cl (&lt; 0.1–3.2 %), Sn (0.3–5.3 %), Zn (0.7–4.5 %), Ce, La and Nb (inclusions)</td><td>Ti (0.2–12 %), Al (&lt; 0.1–7.0 %), Mg (&lt; 0.1–4.4 %)</td></tr></tbody></table>
<figure><img src="image_4.png" /><figcaption>**Fig. 2.** Cytotoxicity induced by urban and rural TRWP of two sizes: 0.39–0.69 µm and 4.2–10.2 µm. Cells were incubated for 24 h with TRWP alone or in the presence of B[a]P 20 µM. For comparison, cells were also incubated with B[a]P alone (20 µM). Relative LDH release was calculated as the ratio of LDH released from treated cells to LDH released by control (unexposed) cells. Results are means of three independent experiments, each performed in quadruplicate. The standard error of the mean is also indicated. C-: negative control: cells alone. C+: positive control: lyzed cells. *** p < 0.001 as calculated compared to C- by a two-way ANOVA analysis and Tukey post-tests.</figcaption></figure>

<figure><img src="image_5.png" /><figcaption>**Fig. 3.** Relative ROS production in response to urban and rural TRWP of two sizes: 0.39–0.69 µm and 4.2–10.2 µm. Cells were incubated for 90 min (A) or 24 h (B) with TRWP alone or in the presence of B[a]P 20 µM. For comparison, cells were also incubated with B[a]P alone (20 µM). Results are expressed as the ratio of ROS produced by treated cells to ROS produced by control cells, and represent the means of three independent experiments, each performed in quadruplicate. The standard error of the mean is also indicated. C-: negative control: cells alone. C+: positive control: cells incubated with 1 µM H₂O₂. *** p < 0.001 as calculated compared to C- by a two-way ANOVA analysis and Tukey post-tests.</figcaption></figure>

proinflammatory response induced by TRWP (according to the trip driven, their concentration, size, and the absence or presence of B[a]P) as assessed by the production of the TNF-α cytokine.

The figure 4 demonstrates that all types of TRWP (urban or rural and in both sizes) were capable of eliciting a highly significant dose-dependent proinflammatory response. A similar observation was made for TRWP in the presence of B[a]P. For samples with TRWP alone, the relative proinflammatory response was highly linear (R² > 0.99), whereas the presence of B[a]P resulted in more irregular or logarithmic evolutions (at least for TRWP collected in urban areas). This was probably due to the B[a]P's greater contribution to the overall proinflammatory response at lower TRWP concentrations.

#### 3.2.3.2. Impact of the TRWP size. 
Fig. 5 compares the proinflammatory effects induced by TRWP of different sizes.

Regarding the role of TRWP size, the fine urban TRWP (0.39–0.69 µm) induced significantly greater proinflammatory effects than the coarse urban TRWP (4.2–10.2 µm). The ratio of the proinflammatory responses (i.e., of the fine vs. coarse urban TRWP) ranged from 1.6 to 2.6, and increased with TRWP concentrations between 18 ± 3 µg/mL and 30 ± 4 µg/mL. Similar increases (i.e., factors ranging from 1.5 to 2.6) were observed in the presence of B[a]P, which highlights its marginal role in modulating local changes in the proinflammatory response to TRWP size. As regards rural TRWP, no direct comparisons could be made due to the lack of common TRWP concentrations. However, coarse TRWP at the
<figure><img src="image_6.png" /><figcaption>**Fig. 4.** Proinflammatory response induced by urban (A, B) and rural (C, D) TRWP of two sizes: 0.39–0.69 µm (A, C) and 4.2–10.2 µm (B, D). Cells were incubated for 24 h with TRWP alone (plain bars) or in the presence of B[a]P (20 µM, hatched bars). For comparison, cells were also incubated with B[a]P alone (20 µM). Results are expressed as the ratio of TNF-α produced by treated cells to that produced by control (unexposed) cells. Results are means of three independent experiments, each performed in quadruplicate. The standard error of the mean is also indicated. C-: negative control: cells alone. * * p < 0.05, ** * * p < 0.001 as calculated compared to C- by a two-way ANOVA analysis and Tukey post-tests. Intergroup comparison: # p < 0.05, ### p < 0.001 as calculated by a two-way ANOVA analysis and Tukey post-tests. ns: non-significant.</figcaption></figure>

<figure><img src="image_7.png" /><figcaption>**Fig. 5.** Proinflammatory response induced by urban (A) and rural (B) TRWP of two sizes: 0.39–0.69 µm and 4.2–10.2 µm after a 24 h cell-particles contact. Results are expressed as the ratio of TNF-α produced by treated cells to that produced by control (unexposed) cells. Results are means of three independent experiments, each performed in quadruplicate. The standard error of the mean is also indicated. C-: negative control: cells alone. * * p < 0.05, ** * * p < 0.001 as calculated compared to C- by a two-way ANOVA analysis and Tukey post-tests. Intergroup comparison: ### p < 0.001 as calculated by a two-way ANOVA analysis and Tukey post-tests.</figcaption></figure>

lower concentrations (9 ± 1 µg/mL and 18 ± 3 µg/mL) showed a proinflammatory response similar to that of fine TRWP at the highest concentrations (60 ± 7 µg/mL and 113 ± 7 µg/mL), suggesting that coarse rural TRWP may be more proinflammatory than fine rural TRWP.

### 3.2.3.3. Impact of the urban vs rural origin of TRWP.
Fig. 6 compares the proinflammatory effect induced by TRWP of similar size but of different origins (urban vs rural).

Based on the comparison of the responses elicited by TRWP at similar concentrations, we observed that while urban fine TRWP induced significantly more TNF-α production than rural fine TRWP (factors ranging from 1.6 to 2.3), coarse urban TRWP induced a significantly decreased TNF-α production compared to coarse rural TRWP (from 0.4 to 0.6). Similar factors (i.e., 1.8–2.0 and 0.4–0.9, respectively) were

found in the presence of B[a]P.

Taken together, these data demonstrate that the size and origin of TRWP (as derived from their formation conditions and chemical composition) are key factors in controlling their proinflammatory response.

## 4. Discussion

Despite the fact that TRWP may not be overly cytotoxic, the possibility of cytotoxicity cannot be dismissed. In fact, Holder et al. (Holder et al., 2012) observed an LDH binding effect with activated carbon, soot, and oxidized soot in the presence of epithelial bronchial cells. Furthermore, TRWP are highly hydrophobic particles that incorporate various mineral and organic matrices, including carbon black as a filler (which
<figure><img src="image_8.png" /><figcaption>**Fig. 6.** Proinflammatory response induced by urban and rural TRWP of two sizes: 0.39–0.69 µm (A) and 4.2–10.2 µm (B) after a 24 h cell-particles contact. Results are expressed as the ratio of TNF-α produced by treated cells to that produced by control (unexposed) cells. Results are means of three independent experiments, each performed in quadruplicate. The standard error of the mean is also indicated. C-: negative control: cells alone. \* *p* < 0.05, \*\*\* *p* < 0.001 as calculated compared to C- by a two-way ANOVA analysis and Tukey post-tests. Intergroup comparison: \*\*\*\* *p* < 0.001 as calculated by a two-way ANOVA analysis and Tukey post-tests.</figcaption></figure>

has a much higher surface-to-volume ratio than soot). Besides, cells incubated with B[a]P alone or in combination with TRWP did not exhibit significant cytotoxicity, which supports the absence of a cumulative or synergistic effect between the two pollutants. Lower LDH concentrations were observed in the presence of TRWP and B[a]P than in the presence of B[a]P alone. These results reinforce the hypothesis of partial LDH binding by TRWP, particularly at higher TRWP concentrations in urban samples.

The data obtained in this study are consistent with previous literature showing that a proinflammatory response is one of the central mechanism of TRWP toxicity (Bouredji et al., 2023). Indeed, airborne tire wear particles have been shown to induce an *in vitro* inflammatory response in various cell models such as human alveolar cells (Gualtieri et al., 2008, 2005a, 2005b), macrophages, nasal epithelial and bronchial epithelial cells (Karlsson et al., 2011, 2006; Lindbom et al., 2007, 2006). It has also been shown that the intensity of the TRWP inflammatory response varies depending on the nature of the road pavement or the type of tire (Lindbom et al., 2007, 2006; Poma et al., 2019). Furthermore, we have previously demonstrated that this intensity is also dependent on TRWP size (Bouredji et al., 2025, 2024a, 2024b).

In this study, we emphasize that the origin of TRWP is an additional factor contributing to their toxicity. This finding is consistent with previous studies and can be explained by the fact that the materials abraded from the pavement, as well as the contaminants from the road surface that are incorporated into the TRWP, vary qualitatively and quantitatively depending on the type of trip (Baensch-Baltruschat et al., 2020; Fussell et al., 2022; Gustafsson et al., 2008; Harrison et al., 2021; Lindbom et al., 2007, 2006; Piscitello et al., 2021). The local mechanisms responsible for TRWP formation can also greatly condition the size and chemical composition of emissions due to the different amounts of heat and friction generated during the tire-road surface interaction. In fact, despite the overall lower braking pressure (average of 6.2 ± 0.2 bar vs. 9.4 ± 0.2 bar), the urban trip is characterized by a significantly longer cumulative duration of braking: equal to 23 % of the travel time when considering braking pressures > 1 bar, against 12 % for the rural trip. This resulted in a 50 % greater loss of weight of the instrumented front driver's side tire (i.e., 29 vs. 20 mg/tire/km) and lower embedding of the abraded tire tread fragments in the rest of the TRWP matrix (Muresan et al., 2024) (Table S1). These fragments favor the release of soluble components from the tire, including antioxidants, kerosene, aromatic oils, etc., that would readily contribute to the *in vitro* or *in vivo* effects of TRWP. Overall, there are likely to be specific characteristics related to the physicochemical properties, toxicological characteristics, and mechanisms of TRWP generation in each region, indicating a limited level of generalizability of the data.

Specifically, we observed that the fine urban TRWP induced

significantly greater proinflammatory effects than the coarse urban TRWP. Various unidentified physicochemical and biological factors may explain this more acute effect. However, it aligns with stronger local emission of tire tread fragments that are free of any mineral matrix or that are present on the surface of the TRWP. These fragments are more likely to release labile compounds into in the cell culture medium. Additionally, the same TRWP exhibit higher contents of Fe and heavy metals (i.e., Cu, Sn and Zn; see Section 3.1), which exhibit substantial cell toxicity and are commonly associated with brake emissions (Banfalvi, 2011; Kumar and Kumaran, 2019). Fine urban TRWP occasionally include Ce, La and Nb and are more proinflammatory than fine rural TRWP, which are comparatively rich in Ti, Al and Mg (Table 1). Overall, the stronger proinflammatory response of fine urban TRWP would reflect the wide variety of pollutants they locally collect. These include a heterogeneous mixture of tire tread fragments, pavement components, and, more importantly, ambient pollutants and other vehicular emissions: such as heavy metals and rare earths that are incorporated by TRWP at the tire-pavement interface, or following their deposition and potential resuspension. Lubrication chemicals can also be present. In this context, Nosratabadi et al. investigated the effect of road and brake wear particles on pulmonary function and biomarkers in isolated perfused rat lungs (Nosratabadi et al., 2023). Thus, improving environmental quality on local and metropolitan scales seems unavoidable to significantly reduce the proinflammatory effects of fine TRWP emissions in urban areas. Although the shift towards electric vehicles is likely to decrease concentrations of exhaust pollutants and brake particle emissions, reducing the intrinsic toxicity of TRWP (i.e., the proinflammatory effects induced by tire and road fragments in TRWP) is challenging due to the limited of knowledge of electric vehicle tire formulations and emission dynamics.

The study of particle size in rural areas supports the idea that the relative proinflammatory response of TRWP is greatly influenced by the pollutants they collect during formation or following emission. In fact, major differences are found between: i) rural and urban fine TRWP in terms of both response and chemical content, and ii) the response and variety of chemical elements observed in rural particles of different sizes. The identified exogenous elements in rural TRWP of different sizes are primarily of terrigenous origin (Fe, Si, Ca, Al), or include moderately and long-term effect toxic metals (Cr, Ti, Mg, Mn). Unlike heavy metals found in urban TRWP fines, these elements hardly affect the intensity of the TRWP inflammatory response, although iron can have some toxicity as described in the literature. Indeed, its toxicity is largely due to its ability to catalyze the formation of radicals (through the Fenton reaction) which can attack and damage DNA and other biomolecules. Overexposure to these effects in the cell promotes cell death and tissue damage and can lead to carcinogenesis (Bystrom et al., 2014; Fontecave
and Pierre, 1993; Papanikolaou and Pantopoulos, 2005; Winterbourn, 1995). As for the intrinsic toxicity of TRWP, the percentage of tire fragments in TRWP increased significantly from large to fine rural TRWP: 1.4 ± 0.2 % vs. 6.1 ± 0.8 % (see Section 3.1). As mentioned earlier, the relative enrichment of fine rural TRWP with tire fragments is likely due to the abrasive action of terrigenous contaminants that deposit on rural roads. The enrichment seems to contradict the lower proinflammatory responses of these particles, which arises from the fact that tire fragments are more deeply embedded within the mineral matrix of rural TRWP (Muresan et al., 2024). This more pronounced embedment is true for both fine and coarse emissions and translates in limited transfer of proinflammatory soluble TRWP compounds (including antioxidants, kerosene, and aromatic oils) into the culture medium. Hence, the greater proinflammatory effects of coarse rural TRWP is very probably associated with unidentified factors.

The unidentified factors may be biological, such as the presence of fungi, viruses, or bacteria in TRWP emissions. They could also be due to plant protection products that were spread in nearby agricultural fields: i.e., coarse particles from treated agricultural soils would settle on the pavement. The latter deposits would have been incorporated into TRWP by mixing with tire tread and road materials at the tire-pavement interface. In fact, the agricultural fields in the traveled region are significant historical reservoirs of xenobiotics, including herbicides, fungicides, and insecticides. Of the hundreds of plant protection products from the chemical industry, the seven most widely sold in France are persulfocarb, glyphosate, pendimethalin, S-metolachlor, chlormequat chloride, fosetyl-aluminum, and chlortoluron. Almost 15 % of them are classified as carcinogenic, mutagenic, and/or reprotoxic (MATTE, 2024). Molecules like glyphosate, S-metolachlor and chlortoluron, as well as their metabolites, are volatile, soluble and/or persistent in soils (Table S2). This makes them conducive to dispersing throughout all environmental compartments and impregnating ecosystems on a regional scale, including as aerosols that can induce cellular toxicity. In fact, of the 125 pesticides tested in rural French homes, which are typically farther from cultivated fields than traveled roads, 97 were measured in recent dust and up to 111 in aged dust (Béranger et al., 2019). Other studies show that plant protection products applied to agricultural fields concentrate in dust collected tens to hundreds of meters from the fields (Hung et al., 2018; Ward et al., 2006). Depending on wind, soil moisture, and nearby vegetation, they can even be detected up to several kilometers from the fields (Gunier et al., 2011; Zaady et al., 2022). Hence, it was hypothesized that the higher proinflammatory responses to coarse rural TRWP partly stemmed from their higher content of agricultural field components. This hypothesis is supported by the significance of local TRWP emissions (average 55 mg/km vs. 40 mg/km in urban areas), which often include large tire fragments coated with terrigenous elements such as Fe, Si, Ca, Mn, and Al (Table S1). It is further reinforced by the fact that the trips took place in November, a traditionally windy month, with historical records showing that gusts exceeded 50 km/h in the days prior. These conditions very likely contributed to the wind erosion of the coarse particles from agricultural fields and their deposition on adjacent roadways: i.e., coarse particles from crop-free fields disperses more broadly in the absence of foliage on nearby hedges and trees (Aveilla et al., 2023).

A limitation of this study is the amount of samples that can be obtained from the collection substrates. This explains why we were not able to perform a large number of analyzes. Therefore, in this first study we focused on screening the basic effects recognized in the literature: cytotoxicity, proinflammatory response and oxidative stress with a limited number of biological endpoints. These preliminary results should not be over-interpreted and further studies are needed to better characterize the proinflammatory response (release of other cytokines, kinetics to determine whether the response is sustained or transient), oxidative stress (triggering of antioxidant systems), etc. It should also be very interesting to study the genotoxicity of TRWP, especially in combination with B[a]P, whose carcinogenic effect is well known. Finally,

the study of leachate and aging of TRWP and their influence on their toxicological effects deserves further interest (Ding et al., 2023).

## 5. Conclusion

A key finding of this study is that fine urban TRWP emissions induce a stronger proinflammatory response than coarse urban or fine rural TRWP emissions. For coarse TRWP, however, rural TRWP emissions induce a stronger proinflammatory response at low dose than fine rural or coarse urban TRWP emissions do. These results demonstrate that the size and origin are both important factors in controlling TRWP proinflammatory response, with origin being closely related to chemical composition. Furthermore, our findings indicate that the TRWP proinflammatory response has at least two sources: one source is intrinsic to the tire tread and pavement fragments in the TRWP, while the other is related to the exogenous components the TRWP collected. The TRWP of various origins likely exhibit a relatively consistent and significant degree of intrinsic proinflammatory response. However, this degree is augmented by the exogenous TRWP components stemming from local road traffic emissions, ambient atmospheric pollution, and road surface contaminants released by human activity near roads. Consequently, this exogenous toxicity varies over time and across territories, necessitating tailored mitigation measures.

## Funding

This work was supported by the Agence de l'environnement et de la maîtrise de l'énergie (ADEME). Grant number: TEZ21-009.

## CRediT authorship contribution statement

**Abderrahmane Bouredji**: Writing – review & editing, Investigation, Formal analysis, Conceptualization. **Laurence Lumière**: Writing – review & editing, Resources, Investigation, Formal analysis. **Riadh Lakhmi**: Writing – review & editing, Supervision, Conceptualization. **Bogdan Muresan-Paslaru**: Writing – review & editing, Validation, Resources, Methodology, Formal analysis. **Jérémie Pourchez**: Writing – review & editing, Supervision, Conceptualization. **Valérie Forest**: Writing – original draft, Visualization, Validation, Supervision, Funding acquisition, Conceptualization.

## Declaration of competing interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Abderrahmane Bouredji reports financial support was provided by The French Agency for Ecological Transition. Valerie Forest reports a relationship with Michelin that includes: funding grants. Bogdan Muresan reports a relationship with Michelin that includes: funding grants. Laurence Lumière reports a relationship with Michelin that includes: funding grants. Jeremie Pourchez reports a relationship with Michelin that includes: funding grants. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.envint.2025.109920.

## Data availability

Data will be made available on request.
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