title: Cytokinesis-block micronucleus assay in WIL2-NS cells: a sensitive system to detect chromosomal damage induced by reactive oxygen species and activated human neutrophils.
abstract: We have developed a method that can detect the DNA-damaging and cytotoxic effects of physiological levels of reactive oxygen species (ROS) and activated human neutrophils. This was achieved using WIL2-NS cells, a human B lymphoblastoid cell line, as target cells and the cytokinesis-block micronucleus (CBMN) assay. With this method, we observed a 4- and a 30-fold increase in the frequency of micronucleated binucleated cells (MNed BNC) when cells were exposed to 10 and 30 microM hydrogen peroxide, for 1 h, respectively. A dose-dependent increase in the frequency of MNed BNC was also detected when cells were exposed to hypoxanthine (HX)/xanthine oxidase (XO), a superoxide generating system: a 50-fold increase in the frequency of MNed BNC was observed at the highest XO dose (12.5 mU/ml). In this CBMN assay, nucleoplasmic bridges (NPB) in BNC and necrotic cells were also readily detected, especially at the higher exposure doses of hydrogen peroxide or HX/XO. When WIL2-NS cells were exposed to neutrophils stimulated with phorbol 12-myristate acetate (PMA) for 1 h, the frequencies of MNed BNC in WIL2-NS cells increased in a dose-dependent manner (30-fold increase at 100 nM PMA) and with an increasing neutrophil:WIL2-NS co-culture ratio. The frequencies of MNed BNC were closely related to the production of ROS, especially hydrogen peroxide, by the neutrophils. Differentiated HL60 cells (DMSO-treated HL60) also produced ROS in response to PMA. In this case, we used a 'Transwell' system to expose WIL2-NS cells to DMSO-treated HL60 cells, because direct contact with DMSO-treated HL60 cells impaired cell division in WIL2-NS target cells. Exposure to PMA-stimulated DMSO-treated HL60 cells resulted in a PMA dose-dependent increase in the frequency of MNed BNC in WIL2-NS cells. MNed BNC frequencies were positively correlated with NPB (r = 0.61-0.93) and necrosis (r = 0.55-0.86) and negatively correlated with nuclear division index (r = -0.72 to -0. 91) in all of the above experiments. These results suggest that the CBMN assay using WIL2-NS cells is a sensitive assay system to examine ROS-induced chromosomal damage and necrosis by activated human neutrophils.
authors.affiliation: CSIRO Health Sciences and Nutrition, PO Box 10041, Adelaide BC, South Australia 5000, Australia.
authors.forename: K
authors.initials: K
authors.lastname: Umegaki
authors.forename: M
authors.initials: M
authors.lastname: Fenech
mesh_terms: D001402:B-Lymphocytes; D002455:Cell Division; D002460:Cell Line; D018922:HL-60 Cells; D006801:Humans; D006861:Hydrogen Peroxide / Q000633:toxicity*; D048629:Micronuclei, Chromosome-Defective / Q000187:drug effects; D015162:Micronucleus Tests; D009153:Mutagens / Q000633:toxicity*; D009336:Necrosis; D018375:Neutrophil Activation; D009504:Neutrophils / Q000187:drug effects / Q000502:physiology*; D017382:Reactive Oxygen Species; D012680:Sensitivity and Specificity; D013481:Superoxides / Q000633:toxicity; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
chemical_list: D009153:Mutagens; D017382:Reactive Oxygen Species; D013481:Superoxides; D006861:Hydrogen Peroxide; D013755:Tetradecanoylphorbol Acetate