Quantification of Radiation-induced DNA Damage following intracellular Auger-Cascades

The aim my PhD study and the topic of this thesis is to investigate the radiotoxicity and the Relative Biological effectiveness (RBE) of intracellular Auger cascades. A special focus is kept on obtaining reliable absorbed dose calculations and using matched dose rate profiles for the Auger exposed cells and cells exposed to the reference radiation. In order to accomplish this, a new experimental model was developed. The Auger cascades were induced by intracellular decays of the electron-capture radionuclide $^{131}$Cs. The use of radiocaesium allowed me to develop new version of the cellular S-values (S$_{C}$-values). The work can be divided into three steps; Examination of the bio-kinetics of the Auger emitter $^{131}$Cs used in the study, calculations of the S$_{C}$-values and finally the measurement of the RBE of intracellular $^{131}$Cs decays, through ƴH2AX and clonogenic cell survival assay. Methods: A series of experiments examining the cellular uptake, release rate and cellular accumulations of $^{131}$Cs in HeLa and V79 cell cultures were performed. The intracellular $^{131}$Cs activity was measured using liquid scintillation counting. The geometry used for the S$_{C}$-values calculations were a confluent cellular monolayer, with the nuclei dispersed within. The height of this cellular monolayer and the size and shape of the nuclei were determined by confocal microscopy for both HeLa and V79 cell cultures. S$_{C}$-values values were obtained for whole cell to nucleus, S$_{C}$(N←CM), with $^{131}$Cs distributed homogeneously throughout the entire cellular monolayer. Monoenergetic electron emission dose kernels for a point source were calculated using the method applied by MIRD for cellular S-values and using Cole´s pseudo stopping power for electrons in water. K- and L-Auger electron energies and intensities (24.6 keV (9.3 %) and 3.43 keV (79.7 %) respectively) with an intensity normalisation factor of 1.17 were used as input for the S$_{C}$ value calculation. To measure the RBE of intracellular $^{131}$Cs decays, confluent cellular monolayers of HeLa and V79 cells were incubated with $^{131}$Cs containing medium. Using the obtained results of the $^{131}$Cs bio-kinetics, and the S$_{c}$-values, dose rate profiles and absorbed doses for the $^{131}$Cs exposed cells could be calculated. HeLa and V79 cell cultures were then exposed to matched dose rate profiles using 137Cs ƴ-rays as the reference radiation. The biological effects of the two exposures were evaluated using ƴH2AX and clonogenic cell survival. Results: $^{131}$Cs was taken up, accumulated and then released by both cell lines. The uptake and release could be described by exponential equations (A = A0 ∗(1−e−t∗kc) and A = A$_{0}$ *(1 - e $^{-t*kc}$) with k$_{c}$ and k$_{out}$ having values of 1/283 min$^{-1}$ (HeLa), 1/204 min$^{-1}$ (V79) and 1/339 min$^{-1}$ (HeLa) , 1/256 min$^{-1}$ (V79) respectively. The cellular uptake of $^{131}$Cs was found to be mediated by the Na$^{+}$/K$^{+}$-ATPase, supporting the hypothesis of a homogenous, intracellular distribution of $^{131}$Cs. The S$_{C}$-values for 29 and 50 different sized nuclei were found to range from 7.78*10-4 to 7.83*10$^{-4}$ Gy/(Bq*Sec)/pL for HeLa nuclei and from 7.45*10$^{-4}$ to 7.63 *10$^{-4}$ Gy/(Bq*Sec)/pL for V79 nuclei. The S$_{C}$-values were shown to be were very robust and almost independent of cellular and nuclear size. A RBE value of 1 was obtained for HeLa cells using ƴH2AX assays. RBE values of 4.5 ± 0.5 and 3.8 ± 0.8 were obtained for HeLa and V79 cells respectively, using clonogenic cell survival. The RBE values obtained in this study are higher than expected for Auger emitters located intracellularly but not directly intercalated to the DNA. Conclusions: The implication of this study is two-fold. The obtained RBE values give further hope for the development of future Auger therapy, as the Auger emitters might not be needed to bind to the DNA in order to achieve high radiotoxicity. At the same time these RBE values should raise concerns about in the ignorance of Auger electrons in dosimetry of diagnostic nuclear medicine procedures, as the risk associated with these might be underestimated. The RBE of Auger emitter decays clearly need to be further investigated. My new experimental method with the robust absorbed dose calculations, the new concept of S$_{C}$-values, and the applicability to most cells types and all Auger emitters with homogeneous intracellular distribution, could be a valuable tool for such investigations.