Exposure to particle debris generated from passenger and truck tires induces different genotoxicity and inflammatory responses in the RAW 264.7 cell line

A number of studies have shown variable grades of cytotoxicity and genotoxicity in in vitro cell cultures, laboratory animals and humans when directly exposed to particle debris generated from tires. However, no study has compared the effects of particles generated from passenger tires with the effects of particles from truck tires. The aim of this study was to investigate and relate the cyto- and genotoxic effects of different types of particles (PP, passenger tire particles vs. TP, truck tire particles) in vitro using the phagocytic cell line RAW 264.7 (mouse leukaemic monocyte macrophage cell line). The viability of RAW 264.7 cells was determined by the 3- (4,5-dimethylthiazol-2-yl) -5- (3-carboxymethoxyphenyl) -2- (4-sulfophenyl) -2H-tetrazolium (MTS) assay following exposure for 4, 24 and 48 hours to different particle concentrations (10 μg / ml, 25 μg / ml, 50 μg / ml, 100 μg / ml). The effects of particles of passenger and truck tires on cell proliferation and genotoxicity were evaluated by means of the cytokinesis-block micronucleus (CBMN) assay following exposure for 24 hours to different particle concentrations (10 μg / ml, 25 μg / ml, 50 μg / ml, 100 μg / ml). In MTS assay, after 24 hours, it was found that PP induced a 30% decrease in metabolic activity at a concentration of 10 μg/ml, while TP caused reductions of 20% and 10% at concentrations of 10 μg/ml and 50 μg/ml, respectively. At 48 hours after the treatments, we observed increased metabolic activity at 50 μg/ml and 100 μg/ml for the PP while only at 50 μg/ml for the TP. The CBMN assay showed a significant increase in the number of micronuclei in the cells incubated with PP in all experimental conditions, while the cells treated with TP showed a meaningful increase only at 10 μg /ml. We utilized the TNF-α ELISA mouse test to detect the production of tumour necrosis factor-alpha (TNF-α) in RAW 264.7 cells. The effect of passenger and truck particles on TNF-α release was evaluated following exposure for 4 and 24 hours.After 4 hours of incubation, the cells treated with PP and TP at 100 μg / ml showed a slight but significant increase in TNF-α release, while there was a significant increase in the release of TNF-α after 24 hours of incubation with both tire samples in the cells treated with 50 and 100 μg / ml PP. The data obtained show a higher cytotoxic, clastogenic/genotoxic and inflammatory effects of passenger compared to the truck tire particles.

多项研究表明，当直接暴露于轮胎产生的颗粒碎屑时，体外细胞培养物、实验动物及人类体内可出现不同程度的细胞毒性与遗传毒性。然而目前尚无研究对比乘用轮胎颗粒与载重卡车轮胎颗粒的生物学效应差异。本研究旨在以吞噬细胞系RAW 264.7（小鼠白血病单核巨噬细胞系）为模型，体外探究两类颗粒——即PP（乘用轮胎颗粒，passenger tire particles）与TP（载重卡车轮胎颗粒，truck tire particles）——的细胞毒性与遗传毒性效应，并分析二者的关联。本研究采用3-(4,5-二甲基噻唑-2-基)-5-(3-羧甲氧基苯基)-2-(4-磺苯基)-2H-四唑鎓（MTS，3- (4,5-dimethylthiazol-2-yl) -5- (3-carboxymethoxyphenyl) -2- (4-sulfophenyl) -2H-tetrazolium）法检测RAW 264.7细胞的活力：将细胞分别暴露于浓度为10 μg/ml、25 μg/ml、50 μg/ml、100 μg/ml的颗粒悬液中，分别于4、24、48小时后检测细胞活力。采用胞质分裂阻滞微核（CBMN，cytokinesis-block micronucleus）分析法，检测细胞暴露于上述梯度浓度颗粒悬液24小时后，乘用轮胎与载重卡车轮胎颗粒对细胞增殖及遗传毒性的影响。在MTS检测中，暴露24小时后发现：10 μg/ml浓度的PP可使细胞代谢活性降低30%；而TP仅在10 μg/ml与50 μg/ml浓度下分别使细胞代谢活性降低20%与10%。处理后48小时，PP组在50 μg/ml与100 μg/ml浓度下均观察到细胞代谢活性升高，而TP组仅在50 μg/ml浓度下出现该现象。CBMN检测结果显示：所有实验浓度下，PP处理组细胞的微核数量均显著升高；而TP处理组仅在10 μg/ml浓度下出现微核数量的显著增加。本研究采用小鼠肿瘤坏死因子-α（TNF-α，tumour necrosis factor-alpha）ELISA检测法，检测RAW 264.7细胞中TNF-α的表达水平；并分别于暴露4小时与24小时后，评估两类轮胎颗粒对细胞TNF-α释放的影响。孵育4小时后，100 μg/ml浓度的PP与TP处理组细胞的TNF-α释放量均出现小幅但显著的升高；而在孵育24小时后，经50 μg/ml与100 μg/ml PP处理的细胞中，TNF-α释放量均出现显著升高。本研究所得数据表明，相较于载重卡车轮胎颗粒，乘用轮胎颗粒具有更强的细胞毒性、致染色体断裂/遗传毒性与炎症效应。