Please use this identifier to cite or link to this item: http://hdl.handle.net/2307/4512
Title: Telomeres and genome stability in irradiated mammalian cells : effect of radiation quality, dose range, and mitochondrial functionality state
Other Titles: Telomeri e stabilità genomica in cellule di mammifero irradiate : effetto del tipo di radiazione, range di dose e della condizione funzionale dei mitocondri
Authors: Nieri, Dino
metadata.dc.contributor.advisor: Antoccia, Antonio
Sgura, Antonella
Keywords: ionizing radations
radiobiology
cytogenetics fish
Issue Date: 25-Feb-2013
Publisher: Università degli studi Roma Tre
Abstract: The most important ionizing radiation (IR) target in eukaryotic cells is the chromosomal asset. The radio-sensitivity of cycling cells, is generally dependent on their damage repair ability and a set of factors which promotes the maintenance of genome integrity. The aim of this work, dealing with several types of IRs and their effects on cycling cell cultures, is manifold: it is a study on the cytogenetic damage of low doses on normal human fibroblasts (i), it is a study addressed to investigate any hypothetic radiation sensitizer role of the dysfunctions of the telomere homeostasis on lymphoblastoid human cells (ii), and finally (iii) it is addressed to highlight the role of the cytosolic reactive oxygen species (ROS) activity and its contribution to the cytogenetic radio-induced damage on normal human fibroblasts pharmacologically treated to this purpose. (i) For a long time the response of a biological system to IR exposure has been supposed proportional to the dose delivery, even the low-dose range, according to the so-called Linear No Threshold Model. Indeed it is a practical and conservative approach due to the general lack of data in that dose range. Based on deterministic data obtained at higher doses, it is suitable as guideline for the risk assessment in a variety of different situations. Things have changed since a set of non-linear trends (§ 1.16) emerged, such as the low-dose hyper-radio-sensitivity/induced-radio-resistance, the bystander effect, and the adaptive response. So far, the cell survival is the most used endpoint to test the occurrence of such non-linear phenomena, in this work instead, cytogenetic skills have used in order to verify hypothetic non-linear behaviors of endpoints such as the chromosomal damage, the telomere length modulation, and the induced genome instability, in dependence on the dose and the type of IR (low- and high- LET). According to the LNTM the fitted functions of the dose-response charts should be strictly monotonic even in the low-dose range, whilst in event of non-linear behaviors, abnormal peaks or flat trends should be reported. We measured the radio-induced damage in two ways: as induction of chromosome exchanges and breakages, through a whole karyotype painting technique (§ 3.1), and as induction of chromatine bridges in anaphase (§ 3.3), through traditional staining. About the induction of such aberrations, the outcome is that no substantial deviation from linearity has been demonstrated. The underlying model is mere probabilistic: the yield and complexity of damage depends only on the dose delivery, the Relative Biological Effectiveness (RBE) of the radiation (fig 14, 15, 18) and the DNA content of the target (fig 16) without hypersensitivity or threshold effects (§ 4.1, 4.2.1). (ii) Coming to the effect of IR exposure on the telomere, to be reminded that it is an important genome stabilizing factor: it avoids the chromosomal termini to be processed as Double Strand Breaks (DSBs), and the chromosome end-end fusion. An effect of IRs on the modulation of telomere length, in terms both of elongation and shortening depending on the radiation type used, has been reported for high doses. Instead, on the basis of our data, such a modulation is missing in the low-dose range (§ 3.2) for all the radiation types tested. Moreover, the telomere does not significantly participate in the genomic instability triggered on by IRs as revealed by the anaphase bridges analysis (§3.3 fig 18). On the basis of this outcome relative to the low-dose range, speculations on a linearity failure are allowed (§ 4.2.1). Ultimately, low doses of IRs induce chromosome instability, but not via the telomere homeostasis perturbation, despite its well-known role played in the chromosome asset destabilization/stabilization. Therefore, we have been persuaded to weight the telomere role in the radio-resistance at doses of X-rays of therapeutic order of magnitude. As the functional telomere is one genome stabilizing factor, IRs are by definition destabilizing agents, able to induce cell inactivation through the genome injury. Moreover, a cell is characterized by a mean telomere length, and a mean spontaneous lack of some telomeres (basal telomere loss frequency). On the aforementioned assumptions, an enhanced radio-sensitivity rationally is expected in case of telomere homeostasis dysfunction. As amid the same cell line the telomere length is spread around a mean value different from cell to cell, then the telomere-related genome stability is supposed to differ from cell to cell (§ 3.4.1), and by hypothesis it may result in a different inherent radio-resistance. We tested this speculation on several monoclonal lymphoblastoid lines: several clones have been isolated, independently cultured and characterized for the telomere functionality and for the radio-resistance. The outcome is that the short telomeres do not confer radio-sensitivity to the line, while a frequency of telomere loss beyond a threshold does (§ 3.4.6 fig 25), thus the telomere dysfunction may be involved in the differential radio-sensitivity of clones obtained from the same population (§ 4.2.2), and shows potential as radio-sensitivity marker. (iii) The mitochondrion is a physiological source of ROS and the main player of their homeostasis, therefore it is supposed to have some indirect involvement in the radio-damage mechanisms. To be reminded in fact that DNA is injured both by direct radiolysis and by means of reactive species released by the water radiolysis. Far to be clear, this role is object of several speculations: for example, irradiated mitochondria are supposed to establish a long-term ROS release, moreover, the mitochondrial metabolism is supposed to amplify the activity of ROS generated by IRs (§ 1.15). The increased ROS activity, generated by mitochondrial impairment, has been related to chromosomal abnormalities, genomic instability, and telomere damage. On the other hand, ROS overproduction, due to the same impairment, paradoxically may be seen as cytoprotective, through engaging countermeasures, as in the so-called adaptive response (§ 1.18). To address the role of mitochondria-related ROS activity in the response to IRs, we pharmacologically set a fibroblast line upset in the mitochondrial functionality, that showed an increase of the basal ROS activity due to ROS homeostasis imbalance (§ 3.5.1). This model, in which mitochondria are turned into ROS diffusers (fig 29), is aimed to report, through the analysis of cytogenetic endpoints, any differential response to the IRs in comparison to a control line, to ultimately verify whether the increased endogenous ROS activity acts additively or synergistically with the exogenous radio-damage. The pharmacological treatment resulted in a basal increased frequency of DSB (§ 3.5.5) and spontaneous chromosomal fragmentation (§ 3.5.7), conversely no damage on the telomere was detected (§ 3.5.6 fig 30, 31). After irradiation, we observed that the cytogenetic damage yield was the sum of the radio-induced and the basal ROS-induced damage via mitochondrial impairment, without synergistic effect. In conclusion, relatively to our data the mitochondrion is not an important player in the damage induction via IRs (§ 4.3).
URI: http://hdl.handle.net/2307/4512
Access Rights: info:eu-repo/semantics/openAccess
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