Contribution to fundamental aspects of biophysics, radiobiology, and modeling of cellular response to low radiation doses

Tese de doutoramento, Engenharia Biomédica e Biofísica, Universidade de Lisboa, Faculdade de Ciências, 2014This thesis aims at studying the direct and bystander effects (observations in which effects of ionizing radiation arise in non-irradiated cells) induced by alpha-particle irradiation, using a Po-210 source, in a human lung adenocarcinoma cells (A549), and at investigating and providing a contribution to the clarification of the protective or potentially detrimental effects of low-dose exposures (< 100 mGy). Besides addressing and analyzing the toxic effects of α-radiation on the A549 cell line the studies undertaken may contribute toward the goal of improving the effectiveness of targeted therapies, with alpha-radiation, such as radioimmunotherapy or Boron Neutron Capture Therapy (BNCT). The work performed consisted, at first, of a dosimetric study in order to determine the average local dose in a cell monolayer, encompassed by experimental and computational studies, followed by a biological study of the direct and bystander effects of α-radiation exposure. The averaged dose rate at the cell monolayer was calculated; through the Linear-Energy-Transfer (LET) value measured using a Passivated Implanted Planar Silicon (PIPS) detector, to be 25 mGy/min. To study the cellular response at low-dose exposures, several studies were conducted using the A549 cell line and a 210Po α-source. Initially, the effects of directly irradiated A549 cells were analyzed through the micronucleus and clonogenic assay. Then, the time and dose dependence of directly and bystander effects in the region of very low doses, was investigated through micronucleus and clonogenic assay. The study encompassed three different cell culture conditions: i) a culture of irradiated cells, ii) a medium transfer culture with non-irradiated cells and iii) a culture with irradiated cells after centrifugation. A dependence on dose and time was observed in both directly irradiated and bystander cells. Taking the advantage of the in situ γ-H2AX technique, the way how the bystander signals released after low-doses of α-radiation are influenced by the number of irradiated cells was studied. Evidence that bystander signals can easily spread through the culture medium was observed. Moreover, the neighboring of irradiated cells differ with the number of irradiated cells, i.e. with a higher number of cells being irradiated, the cellular damage in bystander areas seems to increase. Finally, the gene expression, in GADD45A, BCL2A1 and PTGS2 genes, was assessed with the qRT-PCR technique. This study revealed that all genes are expressed both in directly irradiated and bystander cells, except PTGS2. Moreover, the differences between gene expression levels in both irradiated and bystander cells, for the same dose value, suggest that the mechanisms underlying the response of both types of cells to radiation are different from each other. Summarizing, our results emphasize that the risks attributable to low doses encompass a complex cellular response. The observed hyper-radiosensitivity, time and dose-dependence of the bystander effects and gene expression profiles and the easily spread of bystander signals over the cell culture, raise important questions about the potentially detrimental effects resulting from low dose exposures, which are not included in a simple linear extrapolation from higher dose effects.Esta tese aborda o estudo dos efeitos biológicos, induzidos por exposição a radiação-alfa (α), em células tumorais de pulmão humano (linha celular A549). Foram quantificados os efeitos biológicos em células diretamente irradiadas e em células bystander (efeitos biológicos observados em células não expostas diretamente à radiação), com o objetivo de aprofundar o conhecimento acerca dos efeitos possivelmente protetores ou potencialmente prejudiciais de uma exposição a baixas doses de radiação (<100 mGy). O trabalho desenvolvido nesta tese permite, não só, analisar o efeito tóxico induzido pela radiação em células epiteliais (primeira linha de defesa do organismo) como também contribuir para o estudo da utilização de radiação-α em terapias como a radioimunoterapia ou a terapia por captura de neutrões com boro. Este trabalho consiste, em primeiro lugar, num estudo de dosimetria, com o objetivo de determinar a dose média absorvida por uma monocamada celular, utilizando uma metodologia experimental e uma computacional, seguido de um estudo biológico dos efeitos diretos e dos efeitos bystander induzidos na linha celular A549 após exposição a baixas doses de radiação-α. A taxa média de dose medida na monocamada celular, através do valor da transferência linear de energia (LET) medido por um detector PIPS (Passivated Implanted Planar Silicon), foi de 25 mGy/min. Com o objetivo de quantificar a resposta celular induzida por baixas doses de radiação foram realizados vários estudos, utilizando a linha celular A549 e uma fonte de radiação-α de 210Po. No primeiro estudo, foi quantificada a lesão e a sobrevivência celular em células diretamente irradiadas, através do ensaio de micronúcleos e clonogénico, respetivamente. Em seguida, investigou-se a dependência, com o tempo de incubação e diferentes valores de dose, da lesão e sobrevivência celular na região de doses muito baixas (