Journal Article

Cytokinesis-block micronucleus assay in WIL2-NS cells: a sensitive system to detect chromosomal damage induced by reactive oxygen species and activated human neutrophils

Keizo Umegaki and Michael Fenech

in Mutagenesis

Published on behalf of United Kingdom Environmental Mutagen Society

Volume 15, issue 3, pages 261-269
Published in print May 2000 | ISSN: 0267-8357
Published online May 2000 | e-ISSN: 1464-3804 | DOI: https://dx.doi.org/10.1093/mutage/15.3.261
Cytokinesis-block micronucleus assay in WIL2-NS cells: a sensitive system to detect chromosomal damage induced by reactive oxygen species and activated human neutrophils

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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 μM 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.

Journal Article.  7643 words.  Illustrated.

Subjects: Clinical Cytogenetics and Molecular Genetics ; Genetics and Genomics

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