Aneuploïdia Radioinduïda

Abstract

Ionizing radiation is a mutagenic agent with a well-known clastogenic effect. At DNA level, effects of ionizing radiation can be detected as chromosome reorganizations. Apart from DNA, other cellular elements like those implicated in chromosome segregation, could be affected and originate aneuploidy, which is the condition where the cell or organism chromosome number differs from a multiple exact of the haploid number. We can distinguish two types of aneuploidy: constitutional aneuploidy and non-constitutional aneuploidy. In the first case, all cells of the organism are affected and it is present in miscarriages and birth defects. In the second case, few cells of the organism are affected and it has major importance in tumour cells. In this work, aneuploidy induction and persistence by gamma rays in somatic cells and the sensitivity of different stages of gametogenesis have been analyzed. The analysis of numerical chromosome abnormalities induced in human lymphocytes irradiated in vitro at 1, 2 and 4 Gy was made combining the techniques of cytokinesis-block micronucleus assay and fluorescent in situ hybridization (FISH) using three centromeric specific DNA probes. In this way, the binucleated cells resulting from the first division during the cell culture become included in the same cytoplasm and the distribution of the signals between the nuclei and/or the micronuclei can be evaluated. A dose-dependent significant increase in the micronuclei frequency was observed, which fits to a linear-quadratic curve; this fact indicates that most of them contain acentric fragments resulting from the clastogenic effect of radiation. Related to the mechanisms that originate aneuploidy, a significant increase was observed at 1, 2 and 4 Gy for nondisjunction and at 2 and 4 Gy for the chromosome loss, being higher for nondisjunction. The analysis of aneuploidy induction and persistence was made on splenocytes cell cultures established at 0, 1, 3 and 28 days post-irradiation (dpi) from mice irradiated in vivo at 1 and 2 Gy of gamma rays. The analysis of the binucleated lymphocytes with two subcentromeric specific DNA probes did not show significant increases in nondisjunction frequency at any day or dose studied. For chromosome loss significant increases in the cell cultures established at 0, 1 and 3 dpi were observed, with the characteristic that most micronuclei showed two signals for the same chromosome leaving the main nuclei monosomic. The metaphase analysis of mice lymphocytes irradiated in vivo at 2 Gy showed that 50% of the segregation abnormalities scored in the interphase nuclei analysis would correspond to whole chromosomes, while the remaining 50% would originate from unstable structural chromosomal abnormalities, as dicentric chromosomes and acentric fragments. Finally, we hypothesize that the behavior observed in somatic cells could be different from the behavior in germ cells. For this reason, an analysis of mouse sperm that had been irradiated in the stages of stem cell spermatogonium, spermatocyte I, spermatid and sperm in a testicular and epididimal location was made. Applying FISH with two subcentromeric DNA probes the degree of disomy and of diploidy was assessed. Significant increases of the two types of chromosome abnormalities in sperm irradiated at stem cell spermatogonium, spermatocyte I and spermatid stages were found, due to chromosome malsegregation in the first two stages and to breakage in the labeled region in the last stage.

Date of Award	4 Feb 2008
Original language	Undefined/Unknown
Supervisor	A. Genescà (Director) & Rosa Miró Ametller (Director)