Nucleoplasmic bridges are a sensitive measure of chromosome rearrangement in the cytokinesis-block micronucleus assay

Abstract

We have performed experiments using the WIL2-NS human B lymphoblastoid cell line and primary human lymphocytes to: (i) determine the importance of including measurements of nucleoplasmic bridges (NPB) in the cytokinesis-block micronucleus (CBMN) assay; (ii) provide evidence that NPB originate from dicentric chromosomes and centric ring chromosomes. In addition, we describe theoretical models that explain how dicentric chromosomes and centric ring chromosomes may result in the formation of NPB at anaphase. The results with WIL2-NS showed that it was possible to distinguish genotoxic effects induced by different oxidizing agents in terms of the NPB/micronucleus frequency ratio. The results with lymphocytes indicated a strong correlation: (i) between NPB, centric ring chromosomes and dicentric chromosomes in metaphases (r > 0.93, P < 0.0001); (ii) between micronuclei (MNi), acentric chromosome fragments and acentric ring chromosomes (r > 0.93, P < 0.0001). The dose-response curves with γ-rays were very similar for NPB, ring chromosomes and dicentric chromosomes, as were the dose-response curves for MNi, acentric rings and fragments. However, not all acentric chromosomes and dicentric chromosomes/centric rings were converted to MNi and NPB respectively, depending on the dose of radiation. Preliminary data, using FISH, suggest that NPB often represent DNA from a structural rearrangement involving only one or two homologous chromosomes. The results from this study validate the inclusion of NPB in the CBMN assay which provides a valuable measure of chromosome breakage/rearrangement that was otherwise not available in the micronucleus assay. The CBMN assay allows NPB measurement to be achieved reliably because inhibition of cytokinesis prevents the loss of NPB that would otherwise occur if cells were allowed to divide.