DNA damage and cytotoxicity in type II lung epithelial (A549) cell cultures after exposure to diesel exhaust and urban street particles

Abstract

Background: Exposure to air pollution particles has been acknowledged to be associated with excess generation of oxidative damage to DNA in experimental model systems and humans. The use of standard reference material (SRM), such as SRM1650 and SRM2975, is advantageous because experiments can be reproduced independently, but exposure to such samples may not mimic the effects observed after exposure to authentic air pollution particles. This study was designed to compare the DNA oxidizing effects of authentic street particles with SRM1650 and SRM2975. The authentic street particles were collected at a traffic intensive road in Copenhagen, Denmark.

Results: All of the particles generated strand breaks and oxidized purines in A549 lung epithelial cells in a dose-dependent manner and there were no overt differences in their potency. The exposures also yielded dose-dependent increase of cytotoxicity (as lactate dehydrogenase release) and reduced colony forming ability with slightly stronger cytotoxicity of SRM1650 than of the other particles. In contrast, only the authentic street particles were able to generate 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) in calf thymus DNA, which might be due to the much higher level of transition metals.

Conclusion: Authentic street particles and SRMs differ in their ability to oxidize DNA in a cell-free environment, whereas cell culture experiments indicate that the particle preparations elicit a similar alteration of the level of DNA damage and small differences in cytotoxicity. Although it cannot be ruled out that SRMs and authentic street particles might elicit different effects in animal experimental models, this study indicates that on the cellular level, SRM1650 and SRM2975 are suitable surrogate samples for the study of authentic street particles.

Figure 1

Cytotoxicity of A549 cells exposed to SRM2975 (solid bars), SRM1650 (hatched bars) and ASPM (open bars). The 100% maximum LDH release is obtained by treatment of cell cultures with Triton X-100, whereas the baseline LDH release in untreated cell cultures is 0%. Bars denote the mean ± SEM; (n = 3). * Statistically significant compared to control p < 0.05 (Kruskal-Wallis test). ^# Higher level of LDH release of SRM1650 compared to SRM2975 (p < 0.01, Mann-Whitney U test). ^¤ Higher level of LDH release of SRM1650 compared to SRM2975 and ASPM (p < 0.05, Kruskal-Wallis test).

Figure 2

Colony forming ability expressed as relative to the number of colonies in the control, which are set to 1. The dots represents mean ± SEM; (n = 3). Decreased colony forming ability of cell cultures exposed to SRM2975 (^#) SRM1650 (*) compared to the unexposed cultures (p < 0.05, Kruskal-Wallis test).

Figure 3

8-oxodG formation in calf thymus DNA measured with HPLC/ED after 30 minutes exposure to A) SRM2975 and B) SRM1650 and C) ASPM; ■ filter 1, ▲ filter 2, △ filter 3, ○ filter 4, ◆ filter 5. For SRM2975 and SRM1650 dots represents mean ± SEM; n = 3. The DNA was incubated at 37°C with 5 mM H₂O₂. For ASPM, the DNA was incubated at 37°C with 10 μM H₂O₂.

Figure 4

DNA damage measured by the comet assay in A549 cells exposed to SRM2975 and SRM1650 for 3, 24 or 48 hours. (A) SB, SRM2975, (B) FPG sites, SRM2975, (C) SB, SRM1650 and (D) FPG sites, SRM1650. Mean ± SEM, n = 9. * Dose where all exposure times are statistically significant compared to control (p < 0.01, ANOVA, except for 3 hours exposure to 100 μg/ml where SRM1650 is tested with Kruskal-Wallis test, p < 0.05). ^# Dose where a single time point is statistically significant compared to the respective control (ANOVA): (A) SRM2975, SB, 2.5 μg/ml, 48 h, p < 0.05, (C) SRM1650, SB, 2.5 μg/ml, 3 h, p < 0.05 and SRM1650, SB, 25 μg/ml, 48 h, p < 0.01, (D) SRM1650, FPG, 2.5 μg/ml, 3 h, p < 0.05 and SRM1650, FPG, 25 μg/ml, 24 h, p < 0.05.

Figure 5

Baseline-adjusted SB (A) and FPG sites (B) in A549 cell cultures exposed 24 hours to SRM1650, SRM2975 and ASPM. The data are obtained from figure 4 and table 1. Data points are mean ± SEM (SRMs, n = 9; ASPM, n = 6). There was no significant difference between the particle's ability to induce SB and FPG sites at any doses (p > 0.05, Kruskal-Wallis test).