Micronuclei induced by aneugens and clastogens in mononucleate and binucleate cells using the cytokinesis block assay

Abstract

The human in vitro micronucleus (MN) test has become a fast and reliable assay for mutagenicity testing. Currently, this assay is mostly performed with cytochalasin B, which prevents cytokinesis, resulting in polynucleated cells. The number of nuclei per cell indicates the number of nuclear divisions that have occurred since the addition of cytochalasin B. It is recommended that MN are only counted in binucleated lymphocytes, because these cells have finished one nuclear division. Therefore, almost no attention has been paid to MN in mononucleated cells. However, recent studies have indicated that aneugens, but not clastogens, also induce MN in mononucleates. In order to evaluate mononucleates to distinguish between aneugenic and clastogenic effects, we tested some typical aneugens and clastogens in whole blood lymphocyte cultures of four donors with the cytokinesis block micronucleus (CBMN) assay. Results showed that the aneugens diethylstilbestrol (80 microM), griseofulvin (25 microg/ml) and vincristine sulphate (15 microg/ml) increased MN frequencies in mononucleated and binucleated cells, whilst the clastogens mitomycin C (500 ng/ml), bleomycin (6 microg/ml) and doxorubicin (20 microg/ml) increased MN frequency only in binucleates. We also tested the Y heterochromatin decondensing drug berenil (300 microg/ml). Berenil induced an extremely high number of MN in mononucleated as well as in binucleated cells, indicating an aneugenic action. This was confirmed by centromere labelling. The results suggest that MN in mononucleates may be an interesting additional parameter in the CBMN assay. Future studies should clarify whether the micronucleated mononucleate cells have escaped the cytokinesis block and become polyploid.

Cytogenetic Studies of Berenil in Mice

“…It induced an extremely high number of MN in mononucleated as well as in binucleated cells in human, indicating an aneugenic action. This was confirmed by cytokinesis block micronucleus assay (Rosefort et al 2004).…”

Cytokinesis-block micronucleus cytome assay

“…The frequency of MNi and NBUDs in non-divided mononuclear cells can also be scored if the level of pre-existing DNA damage needs to be measured; however, this approach cannot be used instead of the CBMN Cyt assay, because it does not account for DNA damage accumulated in the bulk of lymphocytes in vivo while circulating in the quiescent phase, it cannot be used to measure NPBs and does not give the same results as scoring MN and NBUDs in BN cells 3,90,91,97 .…”

“…In the case of lymphocytes, it would be preferable to delay the start of the treatment phase to 48 h after mitogen stimulation at which point a large proportion of the mitogen-activated cells would have progressed from G1 to S, G2 and M phases of the cell cycle. By scoring MN in both mononucleated and BN cells, it should be possible to account for MN expressed before Cyt-B block as well as cells with MN that fail to divide and cells that undergo mitotic slippage 91,97 . The suggested protocol also allows for harvesting of cells at the end of the treatment phase before Cyt-B addition if there is a need to test whether the chemical inhibits cytokinesis.…”

Genotoxic effects of anesthetics in operating room personnel evaluated by micronucleus test

“…The slides were coded at the time of preparation and scoring. The frequency of MN was determined in 2000 BN and in 2000 MO lymphocytes per culture [18,21,28,40]. The slides were analyzed using a Carl Zeiss â microscope with an X400 objective.…”

“…However, it was observed that MN in mononucleated (MO) cells, as well as proliferative index (PI), could provide complementary information, since these data may indicate damage that is present in vivo, before cell culture is set up, which makes them interesting for the purpose of biomonitoring [28,40].…”