This 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. The 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. Winter 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. Milan 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.

The Alamar Blue (AB) assay is widely used to investigate cytotoxicity, cell proliferation and cellular metabolic activity within different fields of toxicology. The use of the assay with nanomaterials (NMs) entails specific aspects including the potential interference of NMs with the test. The procedure of the AB assay applied for testing NMs is described in detail and step-by-step, from NM preparation, cell exposure, inclusion of interference controls, to the analysis and interpretation of the results. Provided that the proper procedure is followed, and relevant controls are included, the AB assay is a reliable and high throughput test to evaluate the cytotoxicity/proliferation/metabolic response of cells exposed to NMs.