Title: The Application of Flow Cytometry to Examine Damage Clearance in Stem Cells From Whole-Body Irradiated Mice

The bone marrow contains many types of cells. Approximately 1-2% of these cells are critical for life, these are the so-called ‘bone marrow stem cells’ which divide indefinitely to produce platelets, red blood cells and white blood cells. Death of the bone marrow stem cells results in a diminished ability of the organism to make new blood cell components and can be fatal without medical intervention, such as a bone marrow transplant. Bone marrow stem cells are considered to be particularly sensitive to radiation injury. Therefore, it is important to understand how these cells response to total body radiation exposure and how these cells can be protected from radiation damage. The aim of this project was to determine if these critical cells in the bone marrow are susceptible to short-term and long-term injury after a whole-body exposure to a sub-lethal low dose of ionizing radiation. The overall aims were to determine if the extent of injury produced by the sub-lethal radiation exposure would be cleared from the stem cells and therefore present no long- term genetic risk to the organism, or if the radiation injury persisted and had an adverse long-term consequences for the cell genome. This research question is of interest in order to define the risks to exposed persons after occupational, accidental or terrorism-related sub-lethal low-dose radiation exposures. The novel aspect of this project was the methodology used to obtain the bone marrow stem cell-like cells and examining the outcomes of sub-lethal low-dose radiation in a mammalian animal model. Four radiation treatments were used: single treatments of 0.01Gy, 0.1 Gy, 1 Gy and ten treatments of 0.1 Gy given over 10 days. Bone marrow stem cell-like cells were then harvested 6 hours, 24 hours and 24 days later. The levels of radiation-induced cell death, damage to DNA and permanent changes to cellular DNA were measured in the isolated stem cell-like cells after each radiation treatment and time point and then the results were compared. As expected, the largest radiation dose produced the greatest level of damage but a linear relationship did not exist between cellular effects and radiation dose. The low dose exposures appeared to be more efficient at producing damage than the highest dose when normalized for the initial extent of damage. Additionally, immune stimulation given prior to radiation exposure appeared to protect the critical bone marrow stem cell population from radiation injury. The data suggest that the response of bone marrow stem-cell like cells to radiation injury is dependent on the extent of the initial levels of damage and the effects of total-body low-dose exposures can not be predicted by extrapolating from high dose exposures. This research has provided new information about the radiation sensitivity of bone marrow stem cell-like cells following total-body exposures, and suggests that these critical cells might be more sensitive to radiation than more mature cells in the bone marrow. Further work is need with intermediate radiation doses to confirm this conclusion.