Our understanding of ionizing radiation and its associated biological effects has recently under- gone a paradigm shift from a DNA-centric model to one inclusive of non-targeted effects (NTE), so called for the lack of direct radiation interaction with DNA. Two effects encompassed within the NTE paradigm are termed genomic instability (GI) and bystander effects (BE). GI can be described as an increase in rate of genetic alterations many cell generations after the initial radi- ation exposure. BE can be defined as the manifestation of radiation like effects in un-irradiated cells that have communicated with cells that have been irradiated either through inter-signalling utilising gap junctions or the secretion of a soluble diffusion signalling factor. The exact mechanisms that underlie these processes are still under investigation but a wealth of evidence suggests that a number of mechanisms are involved. These include; cytokine signalling, oxidative stress, inflammation and sub-cellular alterations, in addition to factors such as genetic background and radiation quality/dose. This study was designed to investigate lysosomal involvement in radiation induced NTE, whether it be downstream of one of the above mentioned mechanisms, or independent of their involve- ment. To this end the primary human fibroblast cell line, HF19, was exposed to X-rays at therapeutic and diagnostic doses of 2 and 0.1 Gy respectively. Bystander groups were also established by media transfer techniques. Cells were analysed over the first 24 hours and then at 1 and 20 population doublings, initially for detection of GI and BE and thus confirm the suitability of the system. The lysosomes were then analysed for permeability and their distri- bution within the cell. Oxidative stress was also measured in a bid to correlate this event with lysosomal perturbations. Finally lysosomal contents, in particular DNaseIIα, were analysed for their cellular location along with analysis of nuclear membrane permeability which we surmised would facilitate the redistribution of lysosomal enzymes. The results demonstrated that HF19 cells were susceptible to the induction of GI and BE. The latter was noted within the first hour following irradiation in both 0.1 and 2 Gy bystander groups. High levels of chromosomal instability were also induced in both 0.1 and 2 Gy directly irradiated groups, 1 population doubling after exposure. Chromosomal instability was still noted at 20 population doublings mainly in the 2 Gy although the 0.1 Gy group did show elevated levels. A similar pattern was observed in the bystander group. However we were unable to detect sustained production of the bystander signal at 20 population doublings. Lysosomal properties were also characterised and measured at corresponding time points; large alterations were observed in the first 24 hours following irradiation, furthermore, the lysosomes appeared more permeable at 20 population doublings especially in bystander groups, however, these changes did not correlate with increases in oxidative stress. As a result we further exam- ined cells for changes in the distribution of lysosomal enzymes, in particular DNaseIIα, however no significant changes were observed. Nuclear permeability was additionally investigated as to whether increased permeability facilitated enzyme redistribution; however permeability ap- peared reduced rather than increased. In summary, our investigations have demonstrated and confirmed that HF19 cells are susceptible to the induction of GI and BE following low LET X-ray exposure. The results suggest that the mechanism of radiation induced GI and BE responses can be correlated with alterations in lysosomal membrane permeability which appears independent of oxidative stress. We also demonstrated that if lysosomes are involved in NTE it is unlikely to be through direct action of DNaseIIα but rather from enzymes such as acid sphingomylinase. To conclude, radiation was able to alter lysosomes and nuclear permeability at delayed time points which was correlated with GI , however it appears DNaseIIα is not involved. It also appears that an early effect of the bystander signal may have antioxidant property.
Bright, Scott J.
Supervisors: Kadhim, Munira
Faculty of Health and Life SciencesDepartment of Biological and Medical Sciences
Year: 2013
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