Induction of micronuclei in cultured human lymphocytes treated with vinblastine before and after mitogen stimulation

The analysis of micronuclei (MN) in cultured human lymphocytes can, in principle, detect exposure to clastogens and aneuploidogens alike. As aneuploidogens such as spindle poisons usually act on dividing cells, it is not clear how an in vivo exposure of resting peripheral lymphocytes to an aneuploidogen could be transmitted and expressed as MN in cultured lymphocytes. This question is fundamental in judging if cultured lymphocytes can be used to detect in vivo exposure to aneuploidogens. In the present study, in vivo exposure of resting lymphocytes to an aneuploidogen was simulated in vitro by a 24-h pulse treatment of human lymphocytes with vinblastine sulfate (VBL) before mitogen stimulation, followed by two washes and culture in the presence of phytohemagglutinin (PHA) for 72 h. This treatment protocol did result in an increased MN frequency, but only at the highest concentration of VBL available for analysis (100 ng/ml). A more effective response, with a significant effect already at 40 ng/ml, was obtained when the 24-h pulse treatment was started at 24 h of PHA-stimulated 72-h cultures. Still much lower concentrations of VBL (1 or 2.5 ng/ml) were effective, when the treatment, started 24 h after culture initiation, was continued for 48 or 72 h (respectively) until cell harvest. These results demonstrate that MN induction by VBL depends, as expected, on the duration and timing of exposure, reflecting the availability of dividing cells during the treatment. The positive MN response obtained in the pulse-treated unstimulated lymphocytes may reflect an effect initiated in the resting stage and retained until mitosis or residual VBL left in the cells or in the cell suspension, despite the washes.