The development of a pan-centromeric marker to quantify cellular radiation damage in health individuals and in an HIV cohort

• Main Author: Swanson, Rosemary Veronica
• Format: Others
• Language: en
• Published: 2010
• Subjects: cell damage; radiation
• Online Access: http://hdl.handle.net/10539/8829

MSc (Med), Molecular Medicine and Haematology, Faculty of Health Sciences, University of the Witwatersrand === INTRODUCTION: Ionising radiation can induce DNA damage, in the form of DNA double strand breaks (DSBs), which the affected cell may or may not be able to repair. Micronuclei are indicators of cytogenetic damage, which result from aneugenic (spontaneous loss of chromosomes) or clastogenic (chromosomal fragments) events. The micronuclei may be centromere-positive (CM+MN) for aneugenic events or centromere-negative (CM–MN) for clastogenic events. A pancentromeric marker would help differentiate between CM+MN and CM–MN, especially important among exposures to very low doses of ionising radiation. METHODOLOGY: Micronucleus assays were performed on blood samples collected from healthy donors and HIV+ donors. The blood samples were irradiated at various doses of ionising radiation. Two methods were used to create a pan-centromeric probe. First, the p82H plasmid, which contains centromere specific α repetitive human DNA sequences, was used. Second, human centromeric sequences were amplified using polymerase chain reaction (PCR). In both cases, the pan-centromeric probe was labelled and hybridised using fluorescent in situ hybridisation (FISH) to micronucleus slide preparations from healthy and HIV+ donors. The slides were scored manually and on an automated system, MetaFer®. RESULTS: The p82H probe did not hybridise to any centromeres when FISH was performed, while the synthetic probe made by means of PCR bound to the centromeres of all chromosomes. Henceforth, all experiments were performed with the synthetic pan-centromeric probe. A dose response study was performed on micronucleus slides from healthy donors, from which significant differences in the number of micronuclei and the percentage of centromere-negative micronuclei could be seen between doses. The HIV study involving HIV+ donors and HIV– controls did not yield any significant differences between the two groups. DISCUSSION & CONCLUSION: Combining the micronucleus assay with the pan-centromeric probe greatly improves its sensitivity. The dose response study corroborated previous work performed by Vral et al (1997). Contrary to what was expected and published (Baeyens et al, 2010), no significant differences were observed between HIV+ and HIV- individuals. Issues, improvements and possible future work are discussed.