Gualtieri M et al. (2005), Impact of tire debris on in vitro and in vivo s...

XB-ART-2063

Part Fibre Toxicol 2005 Mar 24;21:1. doi: 10.1186/1743-8977-2-1.

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Impact of tire debris on in vitro and in vivo systems.

Gualtieri M , Andrioletti M , Mantecca P , Vismara C , Camatini M .

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BACKGROUND: 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. METHODS: 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 mug/ml of zinc salt while A549 cells were exposed for 24, 48 and 72 h to 10, 50, 60, or 75 mug/ml of TD extract. X. laevis embryos were exposed to 50, 80, 100, or 120 mug/ml TD extract. RESULTS: 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 mug/ml of TD extract produced 14.6% mortality in X. laevis embryos and 15.9% mortality at 120 mug/ml. Treatment with 80, 100, or 120 mug/ml TD organic extract increased from 14.8% to 37.8% malformed larvae percentages compared to 5.6% in the control. CONCLUSION: 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.

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Species referenced: Xenopus laevis
Genes referenced: grap2 tle5

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	Figure 1. Ultrastructural morphology of TD particles. The morphology of TD particles is shown. A = a SEM image; B = a TEM image; C = a particle viewed with a focused ion beam instrument (FIB); D = FIB inside view of a TD particle.
	Figure 2. EDX Spectrum of TD particle. Microanalysis of a TD particle: a typical fingerprint of tire rubber is shown. This spectrum refers to the particle shown in Fig. 1A. The co-presence of Zn, S and Si is a significant marker to identify TD. Cu derives from the Cu 300-mesh grid used as support for TD particles.
	Figure 3. FTIR Spectrum of TD extract. Soxhlet extracted TD organic compounds analysed by FTIR spectroscopy. The poly-isoprene fingerprint is evident, which confirmed the presence of this rubber compound.
	Figure 4. Accumulation of Zn in HepG2 cells. Zn levels in HepG2 cells exposed to 50 μg/ml ZnS04 7H20 for the time indicated and measured by ICP-AES. Data (ppm/106 cells) represent the mean of at least three replica with the standard deviation. *Significantly different from the control with the Kruskall-Wallis test (p ≤ 0.05).
	Figure 5. A549 cell viability. Percentage of dead A549 cells (evaluated by Trypan Blue exclusion method). Data refer to the mean and standard deviation of at least three independent experiments. The statistically significant differences (p ≤ 0.05) in viable cell number are marked (*).
	Figure 6. A549 cell proliferation. Cell growth curves at 24, 48 and 72 h after TD organic extract treatment at the doses indicated. A decrease of cell proliferation capability is evident, and it is time and dose dependent. Differences between control and treated samples are marked with * (p ≤ 0.05).
	Figure 7. DNA evaluation by comet assay of A549 cells. DNA strand breakage in A549 cells exposed to TD organic extracts. Strand break formation was quantified with comet moment on control and treated samples. Data are mean ± S.D. of three independent experiments and show a dose-dependent increase of comet moment. An * shows significantly different data (p ≤ 0.05)
	Figure 8. Transmission electron microscope of A549 cells. A = control cells present numerous microvilli and the normal cytoplasmic inclusions. B = a vacuolised cytoplasm (arrows) after exposition to TD organic extract. This modified morphology of the cytoplasm is frequent in cells treated with 75 μg/ml TD organic extract at 72 h. (bar = 2 μm).
	Figure 9. 50 g/L TD eluates and FETAX test. Embryotoxic effects of 50 g/L TD on X. laevis embryos, 120 h, stage 47. (** p ≤ 0.01, % mortality compared to control, χ2-test; ## p ≤ 0.01, % malformed larvae compared to control, χ2-test).
	Figure 10. 100 g/L TD eluates and FETAX test. Embryotoxic effects of 100 g/L TD on X. laevis embryos, 120 h, stage 47. (** p ≤ 0.01, % mortality compared to control, χ2-test; ## p ≤ 0.01, % malformed larvae compared to control, χ2-test).
	Figure 11. TD organic extracts on X. laevis development. Trend of mortality and malformed larvae percentage values after X. laevis embryos exposition to TD extracts. (** p ≤ 0.01, % mortality compared to control, χ2-test; ## p ≤ 0.01, % malformed larvae compared to control, χ2-test).