MICRONUCLEI INDUCED BY ALACHLOR, MITOMYCIN-C AND VINBLASTINE IN HUMAN-LYMPHOCYTES - PRESENCE OF CENTROMERES AND KINETOCHORES AND INFLUENCE OF STAINING TECHNIQUE

Antikinetochore antibodies and fluorescence in situ hybridization with an alphoid centromeric probe were applied to the cytokinesis-block micronucleus (MN) assay to study the suitability of these methodologies to detect clastogenic/ aneugenic activity in isolated human lymphocytes. The chemicals selected for this study were the herbicide alachlor, the clastogen mitomycin-C (MMC), and the aneugen vinblastine sulphate (VBL). Futhermore, MN frequencies obtained from slides stained with May-Grunwald-Giemsa (MGG) and with the DNA fluorochrome 4', 6'-diamidino-2-phenylindole (DAPI) were compared to check if the DNA-specific DAPI facilitated a more accurate recording of MN than the unspecific MGG, The results showed that the detection of kinetochores (KC) or centromeres (CM) within MN are equally reliable and sensitive techniques to study the mode of action of clastogenic and aneugenic agents, The comparison of CM and KC detection in control cultures suggested that up to 17% of spontaneous chromosome-containing MN may be due to KC disruption, whereas the majority are caused by dysfunction in other components of the mitotic apparatus, Alachlor (7.5-20 mu g/ml) and MMC (0.6 mu M) acted as pure clastogens without aneugenic activity, inducing exclusively KC- and CM-negative MN. VBL produced primarily KC- and CM-positive MN, in accordance with its known mechanism of action, A comparison between CM and KC data in the VBL treatment suggested that some 7% of KC-containing MN may not be detected by the probe, The frequencies of MN were generally higher in slides stained with DAPI than in those stained with MGG, especially in controls and clastogen-treated cultures, This finding probably reflects an underestimation with MGG of small, light MN indistinguishable from the cytoplasmic background.