INFLUENCE OF CULTURE TIME ON THE FREQUENCY AND CONTENTS OF HUMAN LYMPHOCYTE MICRONUCLEI WITH AND WITHOUT CYTOCHALASIN-B

The effects of culture time (52, 64 and 76 h) and cytochalasin B (Cyt- B, 3 mu g/ml) on the frequency of micronuclei (MN) harbouring whole ch romosomes and acentric fragments was investigated in purified lymphocy te cultures of five nonsmoking male donors aged 41-50 years. Centromer e-positive (C+) MN were identified by fluorescence in situ hybridizati on, using an alphoid DNA oligomer probe (SO-alpha AllCen) hybridizing to all human centromeres. For each culture time, 2000 cells and 60 MN were scored per donor, both with and without Cyt-B, making a total of 60 000 cells and 1800 MN. The frequency of MN and the proportion of C MN were higher at 64 h and 76 h than at 52 h, irrespective of Cyt-B. The culture time-dependent increase in MN frequency was mainly due to C+ MN which were about 1.5-times more frequent at 64 h and 72 h than a t 52 h. The frequencies of C+ MN, expressed per 1000 nuclei, were simi lar with and without Cyt-B, although the prevalence of C+ MN was consi stently about 10 percent units higher in the former type of culture. T his effect was due to a decreased frequency of centromere-negative (C- ) MN in the binucleate cells, possibly reflecting, e.g. increased incl usion of acentric chromosomal fragments within the main nuclei of such cells, enhanced expulsion of C- MN, or selection against binucleate c ells carrying such MN. In conclusion, the present findings indicate th at MN harbouring whole chromosomes become more frequent at long cultur e times with and without Cyt B and that Cyt-B-induced binucleate cells show a reduced frequency of MN containing acentric fragments. Due to the high background of whole-chromosome-containing MN (mean C+ MN prop ortions ranged from 42.3% to 62.7%), it may be recommended that centro meric fluorescence in situ hybridization is routinely applied when lym phocyte MN are used as a biomarker of human exposure to clastogens.