Molecular and celluar assays after radiation exposure : Part 1 : Influence of temperature during irradiation on the level of DNA damage ; Part 2: Comparison of individual radiosensitivity of peripheral blood lymphocytes from prostate cancer patients and healthy donors
Part 1: The level of cytogenetic damage induced by ionizing radiation under in vitro conditions in human peripheral blood lymphocytes is analyzed for the purpose of biological dosimetry and for assessing the intrinsic radiosensitivity of the blood donors. A factor that is often not regarded, but may influence the level of DNA damage is blood temperature during exposure. Hence, this part of the thesis was conducted to analyse the impact of temperature during irradiation of lymphocytes at 0°C and 37°C on the level of DNA damage using micronucleus assay and comet assay. Whole blood cultures were kept at 0°C and 37°C for 15 min before and during exposure to 2 Gy of X-rays. In some experiments isolated PBL were additionally incubated in the presence of 0.5 M DMSO (radical scavenger). A significantly higher level of micronuclei was found when lymphocytes were kept 15 min before and during exposure at 37°C when compared to 0°C. This effect disappeared in the presence of DMSO, what indicates that the observed temperature effect in micronucleus assay is due to the indirect action of radiation. No temperature effect was observed using alkaline and neutral versions of comet assay. Part 2: There is some evidence that approximately 10% of the population show an enhanced intrinsic radiosensitivity of normal tissue and hence have a higher risk for developing side-effects during or after radiotherapy. Moreover, higher cellular radiosensitivity may also indicate cancer susceptibility. For that reason there is a need for a fast and robust test to assess individual cellular radiosensitivity. Hence, the aim of the second part of the thesis was to find out whether PBL from prostate cancer patients (PC) with strong clinical side effects following radiotherapy show enhanced rates of in vitro radiation-induced DNA damage when compared to patients without side effects and healthy age–matched donors. The study included 20 prostate cancer patients without and 20 patients with acute side-effects during and after radiotherapy, as well as 20 healthy age-matched donors. From each donor, blood samples were collected, exposed to a radiation dose of 0.5 Gy or 1 Gy of γ–rays and analysed for the following biological endpoints: the initial level of dsb and the repair kinetics (γ–H2AX–assay), apoptosis (Annexin V/PI–assay) and the induction of chromatid–type chromosomal aberrations (G2–assay). Significant higher chromatid aberration yield was found in prostate cancer patients when compared to healthy donors. No differences were observed between both patients groups in any in vitro assay. Clinical radiosensitivity in vivo assessed on the basis of the EPIC questionnaire correlated with cellular radiosensitivity in vitro assessed on the basis of chromatid aberration 90th cut-off value analysis for 50-62 % of prostate cancer patients. However, based on the results of all chosen assays 6 prostate cancer patients were identified as cellular sensitive, whereof 4 of them were also clinically sensitive.
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