By:
Traimate Sangsuwan, MBW, Stockholm University
Supervisors:
Siamak Haghdoost and Mattias Mannervik, MBW Stockholm University
Committee:
Ulrich Theopold, MBW, Stockholm University
David Berger, Uppsala University
Bo Stenerlöw, Uppsala University

Investigation of radiation-induced genetic and epigenetic effects using cultured cells and Drosophila as experimental models: with focus on dose rate

Abstract
The mechanisms of radiation-induced long-term effects include genetic (mutation) and epigenetic events. In the first article included in the thesis, we have studied the mutagenic effect of chronic low dose radiation focusing on oxidative stress, not direct DNA damage. By measuring a marker of oxidative stress, 8-oxo-dG in the cell culture medium, we showed that low dose rate (LDR) irradiation of the cells in culture induces prolonged oxidative stress that slows down the growth rate. During oxidative stress, free radicals react with DNA, nucleoside triphosphates (dNTPs), proteins and lipids, and modify their structures. An example of a modified nucleoside triphosphate is 8-oxo-7,8-dihydroxy-2’-deoxyguanosine triphosphate (8-oxo-dGTP) which is a mutagenic molecule. The MTH1 and MYH play important roles in preventing mutations induced by 8-oxo-dGTP. MTH1 cleans the cytoplasm from 8-oxo-dGTP and MYH removes 8-oxo-dG that has been incorporated in front of adenine base in the DNA. We established MYH, MTH1 and MTH1/MYH double knock-down cells and exposed them to chronic irradiation. It was found that chronic irradiation-induced point mutations. MTH1 together with MYH plays an important role in protection against mutations induced by modified dNTPs.

To study epigenetic effects, Drosophila embryos were irradiated with different doses at different dose rates and the development process from embryos to adults was followed. The adults that developed from irradiated F0 early stage embryos were followed up to 10 generations (F10). The embryo is a container of highly proliferative cells where different organs are developed through genetically controlled mechanisms. If the process of development is disturbed, it might lead to persistent alteration in the organism. The alteration could be at the level of gene mutation or epigenetic. Epigenetic processes are important during embryo development for maintenance of cell differentiation. The cellular memory and gene regulations are important mechanisms of epigenetic events. We hypothesize that the long-term chronic exposure of embryos to LDR will not reduce their survival but it could induce persistent phenotypes that can pass through generations.

Embryos from wild-type Drosophila melanogaster, w1118 were irradiated at different stages of zygote, blastocyst and during organogenesis with chronic LDR and high-dose-rate (HDR). We investigated the early and late toxicity effects of exposure. Studies at the level of embryo survival clearly showed that the early stage embryo is the most sensitive stage of the embryogenesis. The late post-irradiation effects such as larval movement behavior or pupation height behavior were also investigated. We found that a certain type of depigmentation on males (A5 segment of the dorsal abdomen) can pass through several generations. We will now use molecular approaches to understand the underlying mechanism/s.