Document Type

Theses, Ph.D

Rights

Available under a Creative Commons Attribution Non-Commercial Share Alike 4.0 International Licence

Disciplines

3. MEDICAL AND HEALTH SCIENCES

Publication Details

Successfully submitted for the award of Doctor of Philosophy to the Technological University Dublin, December, 2008.

Abstract

The 'bystander effect' describes radiation-like damage in un irradiated cells, either in the vicinity of irradiated cells or exposed to medium from irradiated cells. This study aimed to further characterize the, as yet poorly understood, mitochondrial response to both direct irradiation and bystander factor(s). Human keratinocyte epithelial and Chinese hamster ovarian cells were exposed to either y radiation or bystander factor(s) at doses of 5mOy, O.50y and 50y and examined 4-96 hours later. DNA damage was determined using deletion analysis, single strand conformation polymorphism analysis, long range PCR, semi-quantitative PCR and quantitative real-time PCR. Mitochondrial mass was determined using MitoTrackelM This study also evaluated irregular mitochondrial function. This was evaluated by determining whole-cell oxygen consumption rates in a Clarke-type oxygen electrode. The activity of individual mitochondrial enzyme complex function of oxidative phosphorylation was also determined and furthermore, the ability of mitochondria to translate their full complement of mitochondrial DNA encoded proteins was evaluated. Deletion analysis identified a novel deletion in the mitochondrial genome as early as 12 hours post direct irradiation. Point mutations were identified in a non-consistent manner in the D-Ioop region of both CHO-Kl and HPV-O mitochondrial DNA and the Cox 11 region of the CHO-Kl mitochondrial DNA. Real time PCR identified a significant increase in mitochondrial genome frequency, while Mitotracker analysis indicated significant increases in mitochondria mass. Long range PCR identified non specific global damage at 96 hours post direct treatment post lower doses of HPV -0 cells. Loss of enzyme function occurred as early as 4 hours post treatment with recovery being observed 12-96 hours in some but not all complexes demonstrating a non-uniform sensitivity to y radiation. Irregular mitochondrial DNA-di rected protein synthesis was also observed, most notably at 96 hours post treatment. Polarography indicated signi fi cantly reduced oxygen consumption rates exposed in CI-IO-K I cells fo llowed by an apparent recovery, most likely due to increases in mi tochondrial numbers_ This study advances the current understanding of short term mitochondrial damage and response post radiation and ' bystander' factor(s) and identifies a need to further evaluate the long term viability of mi tochondria that survive irradiation. The study also highlights that this small, sensitive yet critical organelle may playa significant role in perpetuating the e ffects of radi at ion damage and may provide for first-response biomarkers of very lowlevel exposure.

DOI

https://doi.org/10.21427/D7WG6C

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