Effects of DNA Damage and Oxidative Stress in Human Bronchial Epithelial Cells Exposed to PM2.5 from Beijing, China, in Winter

Abstract

Epidemiological studies have corroborated that respiratory diseases, including lung cancer, are related to fine particulate matter (<2.5 μm) (PM_2.5) exposure. The toxic responses of PM_2.5 are greatly influenced by the source of PM_2.5. However, the effects of PM_2.5 from Beijing on bronchial genotoxicity are scarce. In the present study, PM_2.5 from Beijing was sampled and applied in vitro to investigate its genotoxicity and the mechanisms behind it. Human bronchial epithelial cells 16HBE were used as a model for exposure. Low (67.5 μg/mL), medium (116.9 μg/mL), and high (202.5 μg/mL) doses of PM_2.5 were used for cell exposure. After PM_2.5 exposure, cell viability, oxidative stress markers, DNA (deoxyribonucleic acid) strand breaks, 8-OH-dG levels, micronuclei formation, and DNA repair gene expression were measured. The results showed that PM_2.5 significantly induced cytotoxicity in 16HBE. Moreover, the levels of reactive oxygen species (ROS), malondialdehyde (MDA), and cellular heme oxygenase (HO-1) were increased, and the level of glutathione (GSH) was decreased, which represented the occurrence of severe oxidative stress in 16HBE. The micronucleus rate was elevated, and DNA damage occurred as indicators of the comet assay, γ-H2AX and 8-OH-dG, were markedly enhanced by PM_2.5, accompanied by the influence of 8-oxoguanine DNA glycosylase (OGG1), X-ray repair cross-complementing gene 1 (XRCC1), and poly (ADP-ribose) polymerase-1 (PARP1) expression. These results support the significant role of PM_2.5 genotoxicity in 16HBE cells, which may occur through the combined effect on oxidative stress and the influence of DNA repair genes.

Keywords: DNA damage; DNA repair gene; PM2.5; human bronchial epithelial cells; oxidative stress.

Figure 1

Effects of PM_2.5 on cell cytotoxicity. (A) Analysis of cell viability. 16HBE were treated with 0–400 μg/mL of PM_2.5 for 24 h. (B) Analysis of the level of lactate dehydrogenase (LDH). 16HBE exposed to 67.5, 116.9, 202.5 μg/mL PM_2.5 for 24 h. Data are shown as mean ± standard deviation (SD) (n = 5). * p < 0.05 compared with control. ** p < 0.01 compared with control.

Figure 2

Effects of PM_2.5 on oxidative stress. (A) Detection of ROS production in 16HBE by DCFH-DA probe. 16HBE were exposed to 202.5 μg/mL PM_2.5 for 0–4 h. (B) Analysis of malondialdehyde (MDA). 16HBE exposed to 67.5, 116.9, 202.5 μg/mL PM_2.5 for 24 h. (C) Analysis of glutathione (GSH). (D) Analysis of HO-1 by western blot. β-actin was used as a loading control. Data are shown as mean ± SD (n = 3). * p < 0.05 compared with control. ** p < 0.01 compared with control.

Figure 3

Effect of PM_2.5 on deoxyribonucleic acid (DNA) strand breaks. 16HBE were exposed to 67.5, 116.9, 202.5 μg/mL PM_2.5 for 24 h. (A) The comet analysis of PM_2.5 on DNA fragmentation. (B) The analysis of PM_2.5 on the expression of γ-H2AX by western blot. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. (C) Immunofluorescence of γ-H2AX in 16HBE. Data are shown as mean ± SD (n = 3). * p < 0.05 compared with control. ** p < 0.01 compared with control.

Figure 4

Effect of PM_2.5 on oxidative DNA damage. 16HBE were exposed to 67.5, 116.9, 202.5 μg/mL PM_2.5 for 24 h. The level of 8-OH-dG in 16HBE was detected by enzyme-linked immuno sorbent assay (ELISA) Data are shown as mean ± SD (n = 3). ** p < 0.01 compared with control.

Figure 5

Effect of PM_2.5 on MN. 16HBE were exposed to 67.5, 116.9, 202.5 μg/mL PM_2.5 for 24 h. Data are shown as mean ± SD (n = 3). ** p < 0.01 compared with control.

Figure 6

Effect of PM_2.5 on DNA repair genes. Western blot of 16HBE exposed to 67.5, 116.9, 202.5 μg/mL PM_2.5 for 24 h. (A) Expression of OGG1. (B) Expression of XRCC1. (C) Expression of PARP1. β-actin was used as a loading control. Data are shown as mean ± SD (n = 3). * p < 0.05 compared with control. ** p < 0.01 compared with control.