Impact of tire debris on in vitro and in vivo systems
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It is estimated that over 80% of respirable particulate matter (PM10) in cities comes from road transport and that tire and brake wear are responsible for the 3–7% emission of it. Data on the indicators of environmental impact of tire debris (TD), originated from the tire abrasion on roads, are extremely scarce, even though TD contains chemicals (zinc and organic compounds) which can be released in the environment.
TD particle morphology was analysed with SEM, TEM and FIB instruments. TD eluates and TD organic extracts were tested at dilution series on human cell lines and Xenopus laevis embryos. 50 and 100 g/L TD were used for the eluates obtained after 24 h at pH 3 and the quantity of zinc present was measured with a ICP-AES. Eluates diluted to 1%, 10%, 50% in culture media and undiluted were used on X. laevis embryos in the FETAX test. HepG2 cells were exposed for 24 h to 0.05 – 50 μg/ml of zinc salt while A549 cells were exposed for 24, 48 and 72 h to 10, 50, 60, or 75 μg/ml of TD extract. X. laevis embryos were exposed to 50, 80, 100, or 120 μg/ml TD extract.
The solution of undiluted 50 g/L TD produced 80.2% mortality (p < 0.01) in X. laevis embryos and this toxic effect was three times greater than that produced by 100 g/L TD. Zn accumulation in HepG2 cells was evident after 4 h exposure. A549 cells exposed to TD organic extract for 72 h presented a modified morphology, a decrease in cell proliferation and an increase in DNA damage as shown by comet assay. The dose 80 μg/ml of TD extract produced 14.6% mortality in X. laevis embryos and 15.9% mortality at 120 μg/ml. Treatment with 80, 100, or 120 μg/ml TD organic extract increased from 14.8% to 37.8% malformed larvae percentages compared to 5.6% in the control.
Since the amount of Zn leached from TD is related to pH, aggregation of particles and elution process, the quantity of TD present in the environment has to be taken into account. Moreover the atmospheric conditions, which may deeply influence the particle properties, have to be considered. The TD organic fraction was toxic for cells and organisms. Thus, because of its chemical components, TD may have a potential environmental impact and has to be further investigated.
KeywordsA549 Cell Comet Assay Diesel Exhaust Particle A549 Cell Line HepG2 Cell Line
Frog Embryo Teratogenesis Assay-Xenopus
Diesel Exhaust Particles
Polycyclic Aromatic Hydrocarbon
Focused Ion Beam
Transmission Electron Microscopy
Scanning Electron Microscopy
Inductively Coupled Plasma-Atomic Emission Spectrometry
Human Chorionic Gonadotropin
Fourier Transformed InfraRed analysis
PM of urban areas is a complex mixture of organic and inorganic components .
Both fine (<2.5 μm) and ultrafine (<100 nm) particles and transition metal components are recognized to be responsible in determining toxicity and potential health effects [2, 3]. The respiratory tract is the major site of exposure to PM, which may affect the tracheobronchial tree and the pulmonary alveoli. PM may cause cardiovascular and pulmonary complications and is responsible for increased percentage of lung cancer . PM toxicity is demonstrated by epidemiological studies  and by investigations of laboratory mammals and pulmonary rodent cell lines [5, 6]. Human cell lines were also utilised to study cytotoxicity, genotoxicity and induction of inflammation by soluble and insoluble compounds of PM [7, 8, 9, 10, 11]. PM10 [12, 13], diesel exhaust particles (DEP) [14, 15, 16, 17] and ultrafine particles [18, 19] have received attention and been extensively analysed for their cytotoxicity, while the particulate deriving by tire abrasion on the road received a consideration limited to its impact on the soil, since it releases a large amount of zinc , and on the ambient air for its metal composition [21, 22]. Previously, the presence of latex allergens in tire dust was suggested by Miguel et al. , who outlined that this component could be an important factor in producing latex allergies and asthma symptoms.
Tire debris (TD), generated from tire wear on roads, include particles with a size larger than 7 μm and range up to >100 μm, but also a population of smaller particles (<1 μm) , less than 20% of the total, according to Cadle and Williams . More recently Fauser  examined the TD size distribution and concluded that it is a bimodal histogram with more than 90% by mass (in the range collected, i.e. <20 μm) smaller than 1 μm and the rest are larger than 7 μm. The total concentration of tire particles in a busy city road is in the range 1–10 μg/m3, with a mean value of 2.8 μg/m3 which represents about 5% of the total airborne particles <20 μm. From these data and those of Tappe and Null  and Ntziachristos  it can be calculated that the 5–7% of these particles are in the PM10 size range of the respirable fraction.
Nelson et al.  and Evans  described the deleterious effect of whole tires and scrap on aquatic organisms produced by the release of zinc in the aquatic environment . Moreover, Stephensen et al.  showed that the organic component is also toxic, since PAHs extracted from tire wear induced ethoxyresorufin-O-deethlyase (EROD) activity on fish.
The European Commission  has outlined the environmental risks related to the emission of TD during normal tire wearing, since the contribution of TD to B[a]P on respirable particles is slightly above 5% in comparison to DEP and will increase when diesel-associated emissions are reduced in the near future. Moreover, TD is a source of zinc to the environment [20, 22, 34].
The purpose of this paper is to investigate TD effects on the development of X. laevis, and on human cell lines. In previous studies, we described the shape [35, 36, 37], physical chemical characterization  of TD and preliminary results on their impact on a cell line  and living organisms . Here, we present evidence that both TD eluates and TD organic extracts are highly toxic to living systems.
Michnowicz and Weaks , Evans  and Nelson et al. identified zinc as the major source of eluate toxicity in the aquatic environment. Rubber compounds contain 2% zinc primarily as zinc oxide used as an activator in sulphur vulcanisation. Therefore, a certain quantity of zinc is leached from TD when it rains and when whole tires or scrap are used as water barriers. We analysed the effect of TD eluates on X. laevis embryos. The eluates were obtained by mixing a known quantity of TD in water at pH 3, since typical pH values of acid rain, caused by anthropogenic emissions, may be in the range of 3–5 . Frog Embryo Teratogeneis Assay-Xenopus (FETAX) is a very sensitive aquatic model to assess toxicity and has been used. Moreover, the effect produced by a zinc salt added to the culture medium was evaluated on the human liver cell line HepG2.
TD extract, whose principal chemical compound was poly-isoprene, was tested on the A549 human alveolar cell line, a standardized model to study human lung damage  and on X. laevis embryos.
We present evidences that the toxicity of the eluates is in part related to the presence of Zn, as previously demonstrated by numerous authors on other living systems. Abernathy et al.  presented evidence that toxicity was not completely eliminated by the presence of the divalent chelator EDTA, and they suggested the presence of further toxic organic components. TD extract containing organic chemical compounds was toxic to the A549 human pulmonary cell line at a dose, which may correspond to the quantity present in the air humans inhale daily. Daily ventilator can be 20 m3, and contains 50 μg/m3 PM10 in a polluted urban area. As previously reported 5–7% of PM is TD; thus the supposed quantity of TD inhaled may be 50–80 μg/m3/day.
2.Materials and methods
2.1 TD particle morphology
TD samples were obtained from laboratory degradation tests with a cryo-fracturing technique starting with tire scrap. The morphology of the fine 1–7 μm particles was analysed with a focused ion beam (FIB), and samples were prepared as described previously . Particles were analysed with a scanning electron microscope (SEM) TESCAN and a transmission electron microscope (TEM) JEOL JEM 1220, according to the protocols described previously .
2.2 TD eluates
Eluates were obtained from 50 and 100 g TD placed in 1 L borosilicated glass bottles containing ultra pure water at pH 3. Zinc quantity was measured after 24 h with an Inductively Coupled Plasma-Atomic Emission Spectrometry (ICP-AES) instrument. A 206.200 nm emission wavelength was used to identify Zn ions which ensured that interfering species constituted a negligible part of the response signal. Zn (15.075 ppm, 5.05 ppm and blank) standards in Milli Q water at pH 3 were used for calibration. The bottles were placed in a mechanical shaker at 50 rpm for 24 h. Then different concentrations of the eluate at pH 3, which simulates the lowest pH value of acid rain caused by anthropogenic emissions , were prepared and sterilised. The dilutions were expressed as a percentage by volume. The TD undiluted leachate was defined as a 100% concentration, while the TD 10% leachate was diluted with 90% medium used in the FETAX test (1, 10, 50 and 100% eluate dilution series were used. Solutions were adjusted to the physiological pH of the FETAX medium before adding the salts.
2.3 TD organic extracts
Tire particles were placed in a cellulose extraction thimble and inserted in a Soxhlet assembly fitted with a 250 ml flask, and then 150 ml of dichloromethane was added. Particles were Soxhlet-extracted for 6 h and then either dried on a rotary vacuum evaporator or used for infrared spectroscopy analysis. TD extract was characterized by Fourier Transformed Infrared Spectroscopy (FTIR). Dichloromethane suspended extract was analysed. Briefly, 50 to 100 μl of TD extract were spread over KBr discs and left to dry. The KBr windows were analysed with a FTIR Midac M200 connected to a PC with a spectrum acquisition, storage and elaboration software. The pellet obtained after evaporation was suspended in DMSO at a ratio 10 μg/μl, frozen to -20°C, and used for the test on the A549 cell line and X. laevis embryos.
2.4 TD treatment on in vitro systems
The human lung A549 and the human liver HepG2 cell lines were routinely cultured at 37°C in Opti-MEM medium (Gibco, Life Technologies BRL, USA) supplemented with 10% heat inactivated foetal bovine serum (Gibco Life Technologies BRL, USA) and 1% antibiotics (penicillin\streptomycin) in a humidified atmosphere containing 5% CO2. The A549 cell line was used to test the effect of TD extracts, dissolved in culture medium to the concentrations 10, 50, 60 and 75 μg/ml for 24, 48 and 72 h. A positive control with DMSO at the concentration of 0.75% (v/v) was performed to verify the effect of the solvent alone at 75 μg/ml treatment.
The HepG2 cell line was used to evaluate the potential effect of the zinc salt ZnSO47H2O. Cells, cultured as reported , were exposed for 2, 4 and 24 h to concentrations of Zn ranging from 0.05 to 50 μg/ml, dissolved in culture medium. The intracellular zinc level was directly measured by ICP-AES and expressed as ppm/106 cells.
2.4.1 Cell viability
A549 cell proliferation and viability were evaluated with Trypan Blue (0,4%, Sigma). Cells were seeded in 6-well plates at 1 × 104 cells/well and treated as described in section 2.4. Control and treated samples were analysed with a reverse light microscope by adding 50 μl of Trypan Blue solution to the culture medium.
2.4.2. Comet assay
Alkaline comet assay was performed on A549 cells [44, 45]. Briefly, TD exposed cells were trypsinized and collected. 250 μl of 0.65% normal agarose was dropped onto frosted slides and left to gel. 100 μl of cell suspension was mixed 1:10 with 0.5% low-melting-point agarose, and 100 μl were pipetted onto the slides and allowed to gel; a final layer of 0.5% low-melting-point agarose was then added. The slides were immersed for 1 h at 4°C in the dark in cold lysing solution (2.5 M NaCl, 100 mM EDTA, 300 mM NaOH, 10 mM Tris, 34 mM N-lauroylsarcosine, pH 10); 10% DMSO and 1% Triton X-100 were added just before use. The slides were put in a electrophoresis chamber with 300 mM NaOH, 1 mM EDTA (pH 13.5) for 15 min. Electrophoresis was carried out at 0.8 V/cm for 15 min. The slides were rinsed three times with neutralization buffer (0.5 M Tris, pH 7.4) and stained with 20 μg/ml ethidium bromide. They were scored under a Zeiss Axioplan fluorescent microscope. For each dose, 400 cells/slide were scored and 25 individual comets were recorded for analysis with a self developed image system. The comet length percentage of DNA in the tail and the comet moment, defined as reported previously , were calculated.
2.4.3 Transmission electron microscopy
Control and treated A549 cells, grown in 5.5 cm2 plastic Petri dishes, were rinsed twice with 0.12 M phosphate buffer and fixed for 1 h with a solution containing 4% paraformaldehyde, 2% glutaraldehyde and 0.12 M sodium cacodylate buffer at pH 7.4. The cells were removed and centrifuged at 13.000 rpm for 15 min. The pellets were rinsed in 0.12 M sodium cacodylate buffer, and postfixed for 1 h in 1% osmium tetroxide buffered with 0.24 M sodium cacodylate. The samples were dehydrated and embedded in EPON 812 resin. Sections were stained with uranyl acetate and lead citrate and then examined with a Jeol JEM-1220 electron microscope.
2.5. TD treatment on in vivo systems
2.5.1 Toxicity of TD eluates on X. laevis
Adult X. laevis (Centre d'élevage de Xenopus du CNRS, Rennes, France) were maintained in aquaria at 22 ± 2°C, alternating 12 h light/dark cycles, and fed a semi synthetic diet three times a week. For each bioassay, six females were injected with 900 IU of human chorionic gonadotropin (HCG). About 16 h later, these females were induced to lay eggs in a 140 mm plastic Petri dishes. The eggs were immediately artificially inseminated with a sperm suspension. One minute later, 30 ml FETAX solution  was added to each dish. Successful insemination was detected when all the eggs were oriented with the dark pole side up . Eight hours later (stage 8, blastula) normal cleavage and development were ascertained. Test solutions were administered from stage 8 to stage 47 (free swimming larva), in a thermostatic chamber at 23 ± 0.5°C. The blastulae were exposed to 1, 10, 50, or 100% dilution series of TD eluates. For each female, three Petri dishes were used for the controls and each of the four solutions was tested in triplicate. At stage 47, all the larvae were evaluated for vitality. The surviving larvae were anaesthetized with MS 222 at the concentration of 100 μg/ml for morphological evaluation. For each experimental group, the number of dead embryos and surviving malformed larvae was recorded.
2.5.2 Toxicity of TD organic extracts on X. laevis
X. laevis embryos were exposed to concentrations of 50, 80, 100 and 120 μg/ml TD organic extracts. Testing media were prepared diluting 10 μg/μl TD extract in FETAX solution, to reach the concentrations used.
The toxicity of extracts was examined by exposure of embryos for 120 h. At stage 8, 12 embryos were distributed in 40 mm Petri dishes containing 10 ml control or test solutions. Each experiment was repeated four times. Controls were in duplicate, as the first one was in control solution and the second one in control solution containing 1.2% (v/v) DMSO. This DMSO % produces 3–4% mortality when used in FETAX medium, according to Dresser et al. . At the end of the experiments, the protocol reported in section 2.5.1 was followed.
2.6 Statistical analysis
The experiments with A549 and HepG2 cells were in triplicates and repeated at least three times. A ANOVA model and Kruskall-Wallis test (Statgraphics Plus 5.0 software) were run to detect statistical significant differences between the control and treated sample groups.
The relationship between the TD extract concentration and the percentage of dead or malformed larvae was investigate by the χ2-test and ANOVA model.
3.1 Particle morphology
3.2 Chemical characterization
3.3. In vitro systems
3.3.1 Zinc salt on the HepG2 cell line
3.3.2 TD extracts on the A549 cell line
Cell viability and proliferation
3.4 In vivo systems
3.4.1 TD eluates on X. laevis development
3.4.2 TD extracts on X. laevis development
An increase of malformed larvae percentages was found at 80 and 100 μg/ml (14.8 %; 14.3 %; p < 0.01) compared to the control (5.6%). At concentration 120 μg/ml 37.8% of larvae were malformed. The most frequent malformation was the abnormal gut coiling, often coupled with generalized edema and/or microphthalmia. Some larvae were affected by heavy morphological alterations and so classified as monstrosity.
The distribution of TD particles should be better analysed together with their resuspension properties once deposited on the land surface. At present TD size distribution [25, 26, 38] and chemical composition [20, 26, 38, 50, 51] have received sufficient attention to state that they can have an environmental impact. Actually, TD particles can be identified by inorganic [26, 38, 52] and organic  tracer species. A better understanding of their morphology has recently been obtained using a FIB instrument , which gives further information on the distribution of tire chemical compounds. It has been proven that both organic components [42, 54, 55] as well as inorganic ones [21, 29, 56] affect the environment. In the literature, it is reported that tire-tread formulations historically include about 2.5% Zn . For several decades, tire wear materials have been recognized as a source of Zn in the environment [20, 22, 29, 30], and recently it has been reported  that 10–40% of Zn from soil-applied TD was released in a labile pool. Thus, particular attention has to be devoted to the zinc release, which is a question of serious debate amongst organizations in Europe . The studies of Smolders and Degryse  and Councell et al.  suggest that Zn in rubber tires may be mobile and bioavailable; elevated Zn levels have been demonstrated in leaching experiments using tire rubber  and simulated rainwater [34, 59]. Zn leaching can be augmented indirectly by changing local ambient conditions (e.g., by lowering pH) and it has been demonstrated that tire wear eluates are toxic to species of alga, Daphnia and fish [29, 60]. This toxicity is related to the presence of zinc in the eluates, and the zinc eluted concentration is always higher than that considered harmful to aquatic organisms. Actually maximum permissible Zn concentrations in water, based on toxicity to aquatic organisms, have been set by EPA at 120 μg/l.
Eluates obtained with 50 and 100 g/L TD at a dilution of 50% and undiluted produced high toxicity in X. laevis embryos. Luo et al. showed that zinc chloride LC50 in FETAX was 55 μg/ml. This concentration produced embryotoxicity and teratogenicity, and it is comparable to that present in our eluates. Our results show that 100 g/L TD eluate is less toxic than 50 g/L. Since TD particles tend to aggregate according to their physical properties, aggregated particles expose to the medium a lower surface area than the dispersed ones. Consequently, physical and chemical interactions between particles and medium are decreased, and less quantity of chemicals is leached in the medium.
It is well documented that zinc can affect living organisms, but its effect on cell lines has received scarce attention. We have exposed the HepG2 cell line to the metal salt ZnSO4.7H2O at different concentrations from 0.05 to 50 μg/ml. The 50 μg/ml dose produced a heavy uptake of the metal, and the ICP-AES analysis revealed that the intracellular level of zinc increased from 4 to 24 h of exposure. The analytical procedure used showed a time-dependent accumulation of zinc in cells exposed for 2, 4 and 24 h, leading to a cytotoxic effect. When the intracellular level of zinc exceeds a certain threshold, a number of homeostatic mechanisms take part in regulating the inside distribution by acting on the flux across the plasma membrane, by sequestering the zinc within subcellular compartments , or by synthesising molecules able to bind zinc tightly .
When tire eluates were treated with EDTA by Abernethy et al.  and Gualtieri et al.  only partial removal of zinc toxicity was observed, and this indicated the presence of an additional organic toxicant. In fact, several reports [23, 54, 55, 60] refer to the presence of organic components that can be leached. The TD organic fraction, obtained by extraction in soxhlet apparatus and analysed with FTIR, contained isoprene as the principal chemical compound. Isoprene is an industrial chemical widely used as the basic monomeric unit in natural rubber and the chemical used predominantly in manufacturing of poly-isoprene and various copolymers, such as butyl rubber. Inhalation studies have demonstrated multiple organ tumorigenic effects with this chemical in mice and rats [62, 63]. Isoprene belong to the terpens chemical family, and several studies were conducted on these compounds. Isoprene and chloroprene are listed in the National Toxicology Program's Report on Carcinogens as reasonably anticipated to be a human carcinogen, even though epidemiology data about these two chemicals are not considered adequate to evaluate their potential carcinogenicity in humans. Moreover, it has recently been demonstrated that isoprene forms guanidine adducts , and that methoprene and its degradation products affect X. laevis development .
The interaction between inhaled particles and lung cells is described in literature [66, 67], as well as the correlation between tire particles and the release of latex allergy proteins [23, 68]. These data show that TD can release chemical compounds of its matrix. Recently, it has also been suggested that TD extractable chemicals are endocrine disruptors , which justifies the increasing interest in this pollutant.
We exposed both the A549 cell line and X. laevis embryos to TD extracts. A549 cell proliferation decreased in a time- and dose-dependent manner, and a significant statistical decrease appeared at 24 h and became more evident at 48 and 72 h at the doses tested. To define the role of TD organic extract as an inducer of damage in cellular DNA, we assessed DNA strand breakage by using a comet assay and found that extracts caused strand break formation. It has been shown using the comet assay that insoluble particles, such as carbon black  and quarz , were able to induce strand breaks in A549 cells, probably via intracellular generation of OH. Further investigation is needed to determine if this is the mechanism by which TD organic extracts act. Organic TD treatment greatly modified the morphology of these cells, and apoptotic figures in the nucleus and the cytoplasm were frequent at the highest dose. Moreover, TD extracts produced significant toxicity in X. laevis embryos, since a significant difference in mortality percentages between the control and the highest dose exposed group was present. Moreover an increase of malformed larvae percentages was evident.
Previous work and these results confirm the significant role of zinc in leached TD and the presence of additional organic toxicants. The studies performed have focused their attention on the potential toxic risk to living aquatic organisms from whole rubber tires or scrap. In this study TD has been investigated for its impact on human cell lines and on X. laevis embryos. TD eluates contain zinc, and we have demonstrated that this metal can accumulate in cells, and affect X. laevis embryos. The TD organic extract was toxic to A549 cells and affected cell morphology, cell proliferation and DNA, and produced severe malformations in developing X. laevis embryos. These results contribute to the knowledge of a PM component, which represents a considerable PM10 percentage, which at present is not considered a 'hazardous substance", but it must be taken into account for its potential environmental impact [71, 33]. Moreover, these results strongly stress the need for further investigation into the distribution of TD as well as on its fate in urban areas.
The authors gratefully acknowledge prof. M. Milani for the assistence in FIB technique and dr M. Acciarri for his technical support in SEM analysis.
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