CHAPTER 5: The Cytokinesis-block Micronucleus Cytome Assay in Human Lymphocytes

Micronuclei (MN) are small additional nuclei within human cells that contain chromosome fragments or whole chromosomes that have been excluded from the main nuclei during nuclear division because they could not engage the spindle and segregate properly to the daughter nuclei during the metaphase–anaphase–telophase transition in mitosis. The cytokinesis-block micronucleus (CBMN) assay, which measures MN exclusively in cells that have completed one nuclear division ex vivo or in vitro in cultured lymphocytes, is one of the best validated methods for measuring DNA damage in humans. However, over the past decade it has become increasingly evident that there are other nuclear anomalies that are indicative of other forms of DNA damage, such as nucleoplasmic bridges (NPB), and nuclear buds (NBUD), which originate from asymmetrically rearranged dicentric chromosomes and elimination of amplified DNA, respectively. In addition, the ratio of mono-, bi- and multinucleated cells provides a measure of the cell division rate. Furthermore, cell death can also be measured by enumerating the frequency of necrotic and apoptotic cells. Consequently, the CBMN assay has now evolved into the CBMN-cytome assay, which is the format that captures the full spectrum of the six biomarkers indicated above. This chapter describes the CBMN-cytome assay (including some new additional biomarkers) and its application in human lymphocytes for in vitro and in vivo genotoxicity studies.