DETECTION OF WHOLE CHROMOSOMES IN MICRONUCLEI OF CYTOKINESIS-BLOCKED HUMAN-LYMPHOCYTES BY ANTIKINETOCHORE STAINING AND IN-SITU HYBRIDIZATION

The effect of cytochalasin B (Cyt-B; 3 and 6 mug/ml; for the last 28 h ) on micronuclei (MN) was studied in 72-b purified lymphocyte cultures of three male donors. The frequency of MN was much higher in multinuc leate cells (mean 100 - 204 MN per 1000 cells) than in binucleate cell s (mean 8.2 - 21.0 MN per 1000 cells), tetranucleate cells containing more MN than trinucleate cells. The presence of whole chromosomes in t he MN was studied in two separate experiments by immunofluorescence us ing antikinetochore (CREST) serum and by a centromeric alphoid DNA oli gomer probe (in situ hybridization, ISH). In the tri- and tetra-nuclea te cells produced by Cyt-B, MN were clearly more often kinetochore-pos itive (K+) (mean 82 - 86%) and centromere-positive (C +) (mean 73-83%) than in mononucleate cells of cultures containing no Cyt-B (mean 63% for CREST and 50% for ISH), indicating that most of the excess MN in t he multinucleate cells were due to whole chromosomes. The binucleate l ymphocytes had about as high prevalence of K+ MN (mean 79 - 84%) as th e tri- and tetra-nucleate cells, despite their low MN count. Also in t he ISH analysis, the majority of MN in binucleate cells were positivel y stained (mean 58-62%). If it is assumed that the extra labelled MN a re due to Cyt-B, the present findings suggest that Cyt-B could be resp onsible for approximately 45 - 57% (CREST data) or approximately 17 - 23% (ISH data) of MN in binucleate cells. The presence of whole chromo somes in the majority of human lymphocyte MN is problematic when the a ssay is used for biomonitoring of exposure to clastogens, where the us ually small effects expected may be masked by the high background. The identification of kinetochores or centromeres in MN might help in dea ling with this problem.