Micronuclei in peripheral blood from patients after cytostatic therapy mainly arise ex vivo from persistent damage

The micronucleus test (MNT) is a well-established assay in genotoxicity testing and human biomonitoring. The cytokinesis-block micronucleus test (CBMNT) is the preferred method for measuring MN in cultured human lymphocytes from human subjects exposed to genotoxins. It is, however, unclear to what extent mutagen exposure either leads to the formation of MN already in vivo or to the formation of MN ex vivo during cell culture as a consequence of persisting DNA damage. To address this question, we investigated peripheral blood of 22 patients who had received cytostatic therapies including drugs with clastogenic and aneugenic effects. We also performed the MNT with blood samples from 13 healthy controls without relevant mutagen exposure. The incidence of MN was studied 24, 48 and 72 h after the start of the culture in mononuclear lymphocytes in cultures without cytochalasin B and also at 72 h in binucleated lymphocytes in the standard CBMNT. The mean frequency of binuclear cells with MN in the CBMNT was clearly increased in blood samples from patients (29.3 versus 10.2 per 1000 in controls). In contrast, mononuclear lymphocytes analysed 24 or 48 h after start of the cultures only revealed a marginal increase in MN frequencies in comparison to controls. These results suggest that mutagen exposure in vivo mainly leads to the formation of MN during ex vivo proliferation of lymphocytes as a consequence of persistent damage. Characterization of MN in binuclear lymphocytes from patients by fluorescence in situ hybridization (FISH) with a pan-centromeric probe indicated that MN arose by clastogenic and aneugenic mechanisms. A high portion of MN was relatively large and exhibited several centromere signals. If the results of this study with patients exposed to cytostatic drugs also apply to other kinds of mutagen exposure, increased MN frequencies in the CBMNT can only be expected for exposures leading to persistent damage in peripheral lymphocytes and MN formation during ex vivo lymphocyte culture.