The exposure to high doses of ionizing radiation (>5Gy) is unequivocally associated with increased cancer risk. However, there is substantial experimental evidence showing that in response to low doses of ionizing radiation (LDR: <100mGy), cells and organisms are benefitted with delayed ageing, improved immunity and reduced cancer growth. These intriguing findings have proposed the “Radiation Hormesis” hypothesis. Herein, I studied the senescence effects of LDR exposure to normal human HFL1 cells and examined transcriptional changes. I found that HFL1 cells exposed to 10 mGy of gamma radiation had delayed senescence measured at 12 weeks post-irradiation compared to unirradiated cells. Through qPCR array analysis, I found that genes involved in human cellular senescence functions are differentially regulated in 10 mGy exposed cells at 12 weeks compared to 1-week post-exposure. A nucleolar protein, SIRT7, that belongs to the family of proteins called Sirtuins with known roles in aging, was found to be upregulated transcriptionally in LDR-exposed HFL1 cells. Knocking out SIRT7 protein significantly accelerated senescence in HFL1 cells suggesting a direct role of SIRT7 in the deceleration of senescence and potentially in mediating radiation hormesis. Furthermore, overexpression of the HRAS oncogene strongly accelerated senescence in HFL1 cells through gene expression of cell cycle regulators and checkpoint proteins. Together, my studies revealed that LDR induces unique transcriptional changes resulting in a potentially radio adaptive protective cellular response. I also discuss the HRAS overexpression system as a time-efficient cellular model that could be used to more rapidly study the effect of LDR on senescence using primary cultures.

Humans have evolved under a field of low level radiation, and continue to be exposed to ubiquitous levels from natural and man-made sources including diagnostic radiology. The computerized tomography scan, in particular, plays an important role in the investigation of disease and its use increased dramatically over the years. This raises the concern that elevation in radiation exposure from x-ray modalities may increase an individual's risk for cancer. The purpose of this study is to help address this issue by measuring biological changes in lymphocytes before and after a CT scan. Venous blood was collected from eight prostate cancer patient:> before and after their scan and delivered to McMaster University at room temperature. For the dicentric assay, 0.5 ml whole blood/tube was irradiated with 3 Gy gamma rays using a 0 ;137 source and then incubated at 37°C for 46 hours. Metaphases were scored by microscopy. For apoptosis and y-H2AX, lymphocytes in media were irradiated on ice with 8 Gy and analyzed by flow cytometry. Biological effects in vivo from the CT scan were minimal for all endpoints when averaged between all donors. Overall, there was a high degree of inter-individual variation for each effect, although no correlation was found between dose (dose length product) from CT and apoptosis as well as the induction of yH2AX foci. The adaptive response also showed patient variation, and the frequency of dicentrics was the only endpoint that was lower overall following CT + 3Gy in comparison to 3 Gy alone. This research presents a challenge to current linear models of radiation associated genetic risk, and shows that individuals respond to radiation differently depending on biological factors. / Thesis / Master of Science (MSc)

La vie sur Terre s’est adaptée à un environnement où il y a un faible et persistent bruit de fond radiatif qui interagit avec les cellules. Loin des effets clairement nocifs des radiations à haute dose, il est difficile d’évaluer et de comprendre les impacts des faibles doses de la radioactivité naturelle sur les systèmes vivants. Nous avons tenté d’étudier si le bruit de fond radiatif est un facteur important dans l’évolution, en menant des expériences évolutives identiques avec Escherichia coli au Laboratoire de Physique Corpusculaire de Clermont-Ferrand, et au Laboratoire Souterrain de Modane. Malgré une différence d’un facteur 7,3 entre les taux d’interaction des rayonnements ionisants avec les cellules dans les deux laboratoires, aucune différence significative n’a pu être trouvée dans le fitness compétitif des populations cellulaires évoluées dans chaque laboratoire. Par simulation, nous avons montré que le taux d’interaction entre le bruit de fond radiatif et E. coli est cent fois plus faible que le taux de mutations d’origine endémique, ce qui renforce l’hypothèse que les radiations naturelles ont peu d’effet sur l’évolution. Dans le cadre du projet Geant4-DNA, nous avons développé une application complète de simulation mécanistique des dommages radio-induits à l’ADN, afin d’explorer davantage cette hypothèse. Avec cette application, on a irradié un modèle du génome d’E. coli, montrant que pour l’irradiation par des électrons d’énergies > 10 keV, le rendement des cassures double brin est de 0,006 – 0,010 CDB Gy-1 Mbp-1, selon le modèle de piégeage des radicaux chimiques. Ce résultat est en accord avec des données expérimentales, et souligne plus encore que les radiations ionisantes d’origine naturelle n’ont qu’une contribution mineure aux mutations responsables de l’évolution. / All life on earth has adapted to an environment where there is a small, persistent, radiation background interacting with cells. Unlike evaluating the clearly harmful effects of high radiation doses, understanding the effects of this low persistent radiation dose on living systems is incredibly difficult. We have attempted to study whether background radiation is an important factor in evolution by conducting identical evolution experiments with Escherichia coli in the Clermont-Ferrand Particle Physics Laboratory and the Modane Underground Laboratory. Despite a 7.3 fold difference in the rate of interactions between the radiation background and cells between the two environments, no significant difference was found in the competitive fitness of the cell populations grown at each location. Using simulations, we showed that the rate at which ionising radiation interacts with cells is one hundred times less frequent than E. coli’s mutation rate in our experimental conditions, supporting the contention that natural radiation has no strong evolutionary effect. To further support this conclusion, we developed a mechanistic simulation for DNA damage as part of the Geant4-DNA project. Using this application, we irradiated a model of an E. coli genome, showing that for electron irradiation > 10 keV, the double strand break yield can be reasonably estimated to be between 0.006 – 0.010 DSB Gy-1 Mbp-1, depending upon the modelling of radical scavenging. This is in agreement with experimental data, further highlighting the small role natural ionising radation plays as a cause of mutations.

Patients in the intensive and critical care unit frequently undergo diagnostic radiology procedures such as computed tomography (CT) and X-ray imaging. As these patients often require respiratory assistance and are vulnerable to infection, it is important to understand the potential acute effects of these procedures on the lungs and immune system. The aim of this study was to determine the acute effects of a single clinically relevant low-dose X-ray exposure in order to establish baseline responses in markers of lung injury and immune function in a rodent model. Male Sprague-Dawley rats (200-250 g) were irradiated with 0, 2, 20 or 200 mGy whole-body X-rays in an XRAD 320 irradiator. Markers of lung injury and immune activation in the lungs and spleen were evaluated 0.5, 4, and 24 h post-irradiation to examine the acute stages of the physiological and immunological response. Intratrachaeal lipopolysaccharide (LPS) exposure was used as a positive control model of acute lung injury. Lung injury endpoints included respiratory mechanics, pulmonary oedema, arterial blood oxygenation, histological analysis, and cellular and proteinaceous infiltrate via bronchoalveolar lavage. Immunological measures in the spleen focused on splenocyte proliferation, using the MTS assay and differential cell counts before and after stimulation with LPS or concanavalin A (Con A), as compared to unstimulated cultures. Splenocyte proliferation in response to Con A, but not LPS, was significantly decreased after 200 mGy in vivo X-irradiation (repeated measures two-way ANOVA with LSD post-hoc, p=0.024). There was a non-significant trend towards increased lung tissue resistance after 200 mGy, with no significant effect on pulmonary oedema, cellular or proteinaceous infiltrate, nor other aspects of respiratory mechanics (two-way ANOVA with LSD post-hoc, p>0.05). A clear understanding of these immunological and physiological effects informs the responsible use of medical diagnostic procedures in modern medicine. Establishment of this model for the elucidation of acute immune effects of low-dose radiation will facilitate future work evaluating these parameters in disease models, mimicking patients in intensive care. / Thesis / Master of Science (MSc) / Diagnostic procedures such as computed tomography (CT) and X-ray imaging are a common part of intensive and critical care medicine. Some physicians are concerned that this exposure to diagnostic radiation may negatively affect the health of their patients, who are prone to infection and who often need a machine to breathe for them. In order for doctors to make informed decisions, the possible effects of these levels of radiation must be understood. To improve this understanding, this study looked at the short-term effects of X-ray doses on key organs affected by critical illness, the lungs, and the spleen, which is an important organ of the immune system that helps fight infection. Using an animal model, doses of X-rays in the range of diagnostic radiation (0-200 mGy) were examined and no significant effect on lung health was found. However, the highest dose of X-rays tested, which is greater than that expected for a single CT scan, did have an effect on cells from the spleen. Spleen cells are designed to multiply when they detect various types of infection, so that there are more immune cells to fight that infection. The cells from animals that were given the highest dose of X-rays didn’t multiply as much in response to infective stimulus as those from animals that received lower doses, or no X-rays at all. Overall, it seems that diagnostic radiation doesn’t have an effect in the lungs, but very high diagnostic doses could slightly affect a patient’s ability to fight infection. It is important to remember that patients in critical care are very sick, so doctors have good reason to use diagnostic tools available to them. Missing a diagnosis has major and immediate consequences, which must be balanced against the potential small risks of using radiation to make that diagnosis.

At low doses, relevant to nuclear incidents and accidental releases of radioactivity, the detriment of radiation extends beyond direct effects. This thesis investigates genomic instability, a subclass of non-targeted effects where damage and lethality is transmitted vertically and expressed in the progeny of cells many generations after initial radiation exposure. Through a series of experiments using clonogenic assay of human and fish cell culture, studies described in this thesis describe lethal mutations, hyper radiosensitivity and increased radioresistance – processes involving repair mechanisms that dictate survival in cells exposed to low doses. Further study investigates the difference in the relative biological effect of alpha particle radiation compared to what is expected at high doses. Results demonstrate increased radioresistance in a human cell line while also revealing increased lethality in a fish cell line confirming the need for consideration of dose-dependence as well as variance in behaviors of different cell lines and species. It is hoped the conclusions of this thesis will inspire the creation of protocols with greater attention to the indirect consequences of exposure to radiation at doses relevant to nuclear incidents and accidents. / Thesis / Master of Science (MSc)