In vitro induction of micronuclei in lymphocytes: the use of bromodeoxyuridine as a proliferation marker

Abstract

A simple method to determine the induction of micronuclei in cultured lymphocytes is described as an alternative to the cytochalasin-B method. It is proposed for use in the evaluation of the genotoxic potential of agents in vitro. It allows the recording of events only in the proliferating population of cells and at the same time it eliminates the possibility of recording combined effects with a cytokinesis-blocking agent. 16 μM bromodeoxyuridine (BrdU) was used to label proliferating cells that were treated with colcemid or mitomycin C at different concentrations. A monoclonal antibody against BrdU incorporated in the DNA and a peroxidase-diaminobenzidine brown stain were used to identify those cycling cells in a slide. To obtain the maximum yield of micronuclei, the best time for the addition of bromodeoxyuridine was found to be at 40 h from the initiation of cultures, 8 h before treating cells with the chemicals. Identification of micronuclei was easy, fast and unequivocal. In addition, the formation of structures similar to micronuclei, but that still are part of the nucleus could be observed. It is not clear if these structures are an intermediate stage in the formation of MN, but this methodology provides the possibility of observing and studying them.