Cell cycle alterations induced by urban PM2.5 in bronchial epithelial cells: characterization of the process and possible mechanisms involved

Abstract

BackgroundThis study explores and characterizes cell cycle alterations induced by urban PM2.5 in the human epithelial cell line BEAS-2B, and elucidates possible mechanisms involved.MethodsThe cells were exposed to a low dose (7.5 μg/cm2) of Milan winter PM2.5 for different time points, and the cell cycle progression was analyzed by fluorescent microscopy and flow cytometry. Activation of proteins involved in cell cycle control was investigated by Western blotting and DNA damage by 32P-postlabelling, immunostaining and comet assay. The formation of reactive oxygen species (ROS) was quantified by flow cytometry. The role of PM organic fraction versus washed PM on the cell cycle alterations was also examined. Finally, the molecular pathways activated were further examined using specific inhibitors.ResultsWinter PM2.5 induced marked cell cycle alteration already after 3 h of exposure, represented by an increased number of cells (transient arrest) in G2. This effect was associated with an increased phosphorylation of Chk2, while no changes in p53 phosphorylation were observed at this time point. The increase in G2 was followed by a transient arrest in the metaphase/anaphase transition point (10 h), which was associated with the presence of severe mitotic spindle aberrations. The metaphase/anaphase delay was apparently followed by mitotic slippage at 24 h, resulting in an increased number of tetraploid G1 cells and cells with micronuclei (MN), and by apoptosis at 40 h. Winter PM2.5 increased the level of ROS at 2 h and DNA damage (8-oxodG, single- and double stand breaks) was detected after 3 h of exposure. The PM organic fraction caused a similar G2/M arrest and augmented ROS formation, while washed PM had no such effects. DNA adducts were detected after 24 h. Both PM-induced DNA damage and G2 arrest were inhibited by the addition of antioxidants and α-naphthoflavone, suggesting the involvement of ROS and reactive electrophilic metabolites formed via a P450-dependent reaction.ConclusionsMilan winter PM2.5 rapidly induces severe cell cycle alterations, resulting in increased frequency of cells with double nuclei and MN. This effect is related to the metabolic activation of PM2.5 organic chemicals, which cause damages to DNA and spindle apparatus.

Combined exposure to nano-silica and lead induced potentiation of oxidative stress and DNA damage in human lung epithelial cells

“…Oxidative DNA damage in lung cells has been extensively reported in in vitro and in vivo systems exposed to PM or UFPs (Gualtieri et al, 2012;Longhin et al, 2013;Meng and Zhang, 2007;Oh et al, 2011). Pb, a prevalent toxic metal component in ambient PM, is known to induce oxidative stress and oxidative DNA damage.…”

Particulate matter, air quality and climate: lessons learned and future needs

“…A cytotoxic response was observed for organic nanoparticles generated by laboratory flame and by diesel engines (Manzo et al, 2010;Sgro et al, 2009). Longhin et al (2013) reported the correlation of OC and DNA damage in an urban environment. OC correlated with oxidative potential in the Long Beach harbour area and in traffic-exhaust-dominated areas (Gualtieri et al, 2011;Verma et al, 2011).…”

Toxicity Research of PM2.5 Compositions In Vitro

“…Longhin et al examined the role of PM 2.5 organic fraction versus washed PM on the cell cycle alterations, and the results have shown that the metabolic activation of PM 2.5 organic chemicals cause damages to DNA and the spindle apparatus, and G2/M arrest and augmented ROS formation follow it, while washed PM had no such effects [30]. …”