Developments of the cytochalasia-b micronucleus/kinetochore assay with Chinese hamster fibroblast cells in vitro

• Main Author: Lynch, A. M.
• Published: Swansea University 1991
• Subjects: 571.6
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.637963

The principal aim of the research described in this thesis was to develop and validate an <i>in vitro</i> micronucleus assay in low passage Chinese hamster Luc2 cells capable of detecting numerical and structural chromosome changes. Chromosome loss was inferred by indirect visualisation of human CREST antikinetochore antibodies bound to centromeres in chemically-induced micronuclei of cytochalasin-B arrested binucleate cells. Ten core chemicals were selcted due to their known or suspected effects on components of the cell division apparatus. These chemicals were colchicine, vinblastine, pyrimethamine, diazepam, chloral hydrate, thiabendazole, hydroquinone, econazole nitrate, thimerosal and cadmium chloride. In addition, 5-Azacytidine was selected to determine if disruption of the centremere would inhibit kinetochore visualisation. Three food additives were also selected for study in the micronucleus kinetochore assay to detect indirect acting mutagens with the use of an external metabolic activation system. Six of the core chemicals induced micronuclei in Chinese hamster Luc2 cells. All six chemicals increased levels of micronuclei which were positive for kinetochore antibody labelling and hence chromosome loss. Similar results were also seen with 5-Azacytidine. The results of these studies show that the cytochalasin-B Mn/k assay is a cost effective, simple and rapid alternative to classical cytogenetic assays for the detection of chemically induced aneuploidy. Future work requires the development of a permanent staining technique to visualise kinetochores and greater standardisation between assay protocols published in the literature. These problems need to be resolved before the assay is accepted by regulatory bodies for routine testing of environmental chemicals.