Clastogenic effects induced in vitro by CCNU and AMD during G2-phase in bone marrow cells of Wistar rats

Three concentrations of CCNU and AMD (1, 5 and 10 micrograms/ml) were given to bone marrow cells of Wistar rats immediately after their removal from the body into the culture medium. Abnormal metaphases observed 2, 3 and 4 h later were considered to represent the effect induced by the two drugs on G2-phase chromosomes in a cell population that traversed in vivo previous phases of the cell cycle. In order to determine if the cells examined at metaphase completed S-phase in vivo, BroU at 100 micrograms/ml was added into the culture medium and metaphases displaying replication banding were scored 2, 3 and 4 hours afterwards. Only 4% of metaphases with incorporated BrdU (Bromodeoxyuridine) were seen after 4 hours exposure and none after 2 or 3 hours, indicating that treatment was given to cells during G2-phase. A dose-related clastogenic action was observed both after AMD and CCNU treatments, but the response was essentially determined by the lapse of time between treatment and mitosis. The highest chromosome damaging effect was induced by CCNU when administered 4 hours before mitosis, while AMD reached the highest clastogenic effectiveness when given 2 hours before mitosis. These results express G2 sensitivity of bone marrow cells that traversed G1- and S-phases in vivo and consequently possessed a biochemical "machinery" initiated and effective into the rat body. The capability of primary cultures of bone marrow cells to mimic the mechanisms of the in vivo clastogenic action is discussed.