Rat Data

Abstract Introduction Human DNA is damaged up to one hundred thousand times per day through exposure to DNA damaging agents such as reactive oxygen species, radiation, chemicals and endogenous metabolic by-products. Defective or inefficient repair of the most lethal kind of DNA damage, DNA double strand breaks which affect both strands of the DNA has been linked with early- onset cancer syndromes, advanced ageing and other neurological and immunological conditions. In Australia, current cancer risk screening is population-based for target groups at risk of breast, bowel and cervical cancer. For individuals identified as having an increased risk of a familial cancer syndrome such as Hereditary Breast and Ovarian Cancer Syndrome, genetic screening is available. However, population-based screens and genetic screening cannot predict the age of onset of disease or risk of early-onset disease. Furthermore, as only 5-10% of all diagnosed cancer cases are linked to hereditary cancer predisposition syndromes, there is a gap in cancer risk screening for individuals that do not have a hereditary cancer predisposition syndrome and do not fit the criteria for population-based screening. This PhD investigated the potential use of the DNA double strand break marker, -H2AX, for individualised assessment of DNA double strand break repair for its potential use in future clinical cancer risk screening. Methods This PhD thesis assessed DNA double strand break damage and repair in three experimental models. The first, a Wistar rat model which is examined in Chapter 4, investigated the link between DNA damage and repair within three haematopoietic tissues: bone marrow, peripheral blood mononuclear cells and spleen. This chapter also explored how ageing influences DNA damage and repair using sibling rats that had their samples analysed at ages 3 months and 15 months. This model also investigated the influence of biological sex on DNA damage and repair. The second study (Chapter 5) explored DNA damage and repair capacity collected from 45 healthy human participants ranging from ages 18-75. The third study (Chapter 6) was a pilot experiment that assessed how a previous history of cancer influences DNA damage and repair using the same series of techniques. The novel factor within this study is the inclusion of DNA double strand break assessment at three-time points; an initial baseline, after two-hours of DNA damaging agent (etoposide) treatment and following a one-hour post etoposide wash-out. All three models used flow cytometry assessment of staining of DNA double strand breaks with - H2AX antibody. Phosphorylation of the histone protein complex from H2AX to -H2AX was labelled with the fluorophore Alexa Fluor 488 and quantified via flowcytometry using gates that detected cells positive for -H2AX labelling. Quantification was calculated by the mean fluorescence intensity of -H2AX and percentage change in the mean fluorescence intensity of -H2AX. Results This research found that -H2AX has potential for use clinically for the assessment of DNA double strand break damage repair. This research also determined a standardised method for the assessment of DNA double strand break repair within bone marrow, peripheral blood mononuclear cell (PBMC)s and spleen and included verification of results in PBMC studies across specimens. Through optimisations and validation of methodology, this research found that for successful assessment of DNA double strand break repair assessment in primary cell culture, cell cultures must be treated with a proliferation stimulant for 72 hours prior to DNA damage induction with a chemotherapeutic DNA damaging agent. This research also found that the optimal time for DNA double strand break repair assessment is one-hour following a two-hour treatment with etoposide. In the Wistar rat model, advanced age was linked to a significantly increased mean fluorescence intensity of -H2AX within baseline sampling of bone marrow and PBMCs. Significant retention of -H2AX was also seen following a one-hour fresh media incubation in bone marrow samples. Non-significant increases in -H2AX following a one-hour fresh media incubation were also observed with advanced age in PBMCs, which demonstrated translational potential for use within human studies. The human model of DNA double strand break damage and repair within PBMCs, found that males had a significantly higher mean fluorescence intensity of -H2AX following a two-hour incubation with etoposide when compared to females in the test cohort (p