title: Cell cycle alterations induced by urban PM2.5 in bronchial epithelial cells: characterization of the process and possible mechanisms involved.
abstract: BACKGROUND
  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.
  
  
  METHODS
  The cells were exposed to a low dose (7.5 μg/cm(2)) 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 (32)P-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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Environmental Sciences, POLARIS Research Centre, University Milano-Bicocca, Piazza della Scienza 1, 20126 Milano, Italy. e.longhin1@campus.unimib.it.
authors.forename: Eleonora
authors.initials: E
authors.lastname: Longhin
authors.forename: Jørn A
authors.initials: JA
authors.lastname: Holme
authors.forename: Kristine B
authors.initials: KB
authors.lastname: Gutzkow
authors.forename: Volker M
authors.initials: VM
authors.lastname: Arlt
authors.forename: Jill E
authors.initials: JE
authors.lastname: Kucab
authors.forename: Marina
authors.initials: M
authors.lastname: Camatini
authors.forename: Maurizio
authors.initials: M
authors.lastname: Gualtieri
mesh_terms: D000393:Air Pollutants / Q000633:toxicity*; D015153:Blotting, Western; D001980:Bronchi / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D002453:Cell Cycle / Q000187:drug effects*; D059447:Cell Cycle Checkpoints / Q000187:drug effects; D002460:Cell Line; D004249:DNA Damage; D004847:Epithelial Cells / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D005434:Flow Cytometry; D006801:Humans; D007150:Immunohistochemistry; D007558:Italy; D048629:Micronuclei, Chromosome-Defective / Q000139:chemically induced; D008856:Microscopy, Fluorescence; D008938:Mitosis / Q000187:drug effects; D010316:Particle Size; D052638:Particulate Matter / Q000633:toxicity*; D017382:Reactive Oxygen Species / Q000378:metabolism; D012621:Seasons; D008941:Spindle Apparatus / Q000187:drug effects / Q000378:metabolism; D057891:Tetraploidy; D014507:Urbanization
chemical_list: D000393:Air Pollutants; D052638:Particulate Matter; D017382:Reactive Oxygen Species