Toxicological Profile of PM from Different Sources in the Bronchial Epithelial Cell Line BEAS-2B

Abstract

The toxicity of particulate matter (PM) is strictly associated with its physical-chemical characteristics, such as size or chemical composition. While these properties depend on the origin of the particles, the study of the toxicological profile of PM from single sources has rarely been highlighted. Hence, the focus of this research was to investigate the biological effects of PM from five relevant sources of atmospheric PM: diesel exhaust particles, coke dust, pellet ashes, incinerator ashes, and brake dust. Cytotoxicity, genotoxicity, oxidative, and inflammatory response were assessed in a bronchial cell line (BEAS-2B). BEAS-2B cells were exposed to different concentrations (25, 50, 100, and 150 μg/mL medium) of particles suspended in water. The exposure lasted 24 h for all the assays performed, except for reactive oxygen species, which were evaluated after 30 min, 1 h, and 4 h of treatment. The results showed a different action of the five types of PM. All the tested samples showed a genotoxic action on BEAS-2B, even in the absence of oxidative stress induction. Pellet ashes seemed to be the only ones able to induce oxidative stress by boosting the formation of reactive oxygen species, while brake dust resulted in the most cytotoxic. In conclusion, the study elucidated the differential response of bronchial cells to PM samples generated by different sources. The comparison could be a starting point for a regulatory intervention since it highlighted the toxic potential of each type of PM tested.

Keywords: BEAS-2B; brake dust; coke dust; diesel exhaust particle; genotoxicity; in vitro; incinerator ash; oxidative stress; pellet ash.

Figure 1

Evaluation of cell viability through Annexin V test in flow cytometry. BEAS-2B cells were treated with 25, 50, 100, or 150 µg/mL of DEP, C, PA, IA, or BD for 24 h. The graph shows the percentage of necrotic cells population, evaluated via Annexin V test in flow cytometry (positive to both PI and Annexin V staining). Results are expressed as mean ± SEM, n = 3. Dashed line represents the mean of the control sample data. Statistical analysis was performed using One-Way ANOVA with Dunnett’s post hoc analysis. * p < 0.05, ** p < 0.01 vs. C-.

Figure 2

Time course of ROS production following PM exposure. Cells were treated with 25, 50, 100, or 150 µg/mL of DEP, C, PA, IA, or BD for 30 min (A) and 1 h (B). Results are expressed as the mean of fluorescence ± SEM, in percentage compared to C- (fixed at 100%, dashed line), n = 3. Statistical analysis was performed using One-Way ANOVA with Dunnett’s post hoc analysis. * p < 0.05, ** p < 0.01 vs. C-.

Figure 3

Quantification of DSBs through γ-H2AX evaluation via immunofluorescence. Cells were treated with 25, 50, 100, or 150 µg/mL of DEP, C, PA, IA, or BD for 24 h. The results are expressed as a percentage of cells classified based on the amount of DNA damage in 3 populations: 0–5 foci, absence of damage, 6–10 foci, medium damage, >10 foci, and high damage (shown in the graph). Dashed line represents the mean value of the control. Results are expressed as the mean of percentages ± SEM, n = 3. Statistical analysis was performed using One-Way ANOVA with Dunnett’s post hoc analysis. * p < 0.05, ** p < 0.01 vs. C-.

Figure 4

Micronuclei evaluation via immunofluorescence. Cells were treated with 25, 50, 100, or 150 µg/mL of DEP, C, PA, IA, or BD for 24 h. Control sample mean is also reported as a dashed line. Results are expressed as the mean of percentages ± SEM, n = 3. Statistical analysis was performed using One-Way ANOVA with Dunnett’s post hoc analysis. * p < 0.05, ** p < 0.01 vs. C-.

Figure 5

Secretion of IL-8. Cells were treated with 25, 50, 100, or 150 μg/mL of DEP, C, PA, IA, or BD for 24 h. Dashed line represents the mean of the control sample. Results are expressed as the mean of pg/mL ± SEM, n = 4. Statistical analysis was performed using One-Way ANOVA with Dunnett’s post hoc analysis. * p < 0.05, ** p < 0.01 vs. C-.