Abstract
DNA double strand break (DSB) formation induced by ionizing radiation exposure is indicated by the DSB biomarkers γ-H2AX and 53BP1. Knowledge about DSB foci formation in-vitro after internal irradiation of whole blood samples with radionuclides in solution will help us to gain detailed insights about dose-response relationships in patients after molecular radiotherapy (MRT). Therefore, we studied the induction of radiation-induced co-localizing γ-H2AX and 53BP1 foci as surrogate markers for DSBs in-vitro, and correlated the obtained foci per cell values with the in-vitro absorbed doses to the blood for the two most frequently used radionuclides in MRT (I-131 and Lu-177). This approach led to an in-vitro calibration curve. Overall, 55 blood samples of three healthy volunteers were analyzed. For each experiment several vials containing a mixture of whole blood and radioactive solutions with different concentrations of isotonic NaCl-diluted radionuclides with known activities were prepared. Leukocytes were recovered by density centrifugation after incubation and constant blending for 1 h at 37°C. After ethanol fixation they were subjected to two-color immunofluorescence staining and the average frequencies of the co-localizing γ-H2AX and 53BP1 foci/nucleus were determined using a fluorescence microscope equipped with a red/green double band pass filter. The exact activity was determined in parallel in each blood sample by calibrated germanium detector measurements. The absorbed dose rates to the blood per nuclear disintegrations occurring in 1 ml of blood were calculated for both isotopes by a Monte Carlo simulation. The measured blood doses in our samples ranged from 6 to 95 mGy. A linear relationship was found between the number of DSB-marking foci/nucleus and the absorbed dose to the blood for both radionuclides studied. There were only minor nuclide-specific intra- and inter-subject deviations.
Highlights
DNA double-strand breaks (DSBs) are critical cellular lesions that can result from ionizing radiation exposure [1] and through other DSB-inducing cytotoxic agents [2,3]
One of the earliest events after DSB formation is the phosphorylation of the histone H2 variant H2AX which is called γ-H2AX since it was first observed in cells irradiated with gamma-rays [1,5,6]
The aim of this study was to develop a method that allows generating a calibration curve for the DSB focus assay after internal irradiation with radionuclides by creating a low dose and low dose-rate blood irradiation situation in-vitro, at dose rates that are similar to the ones that have been observed in nuclear medicine patients
Summary
DNA double-strand breaks (DSBs) are critical cellular lesions that can result from ionizing radiation exposure [1] and through other DSB-inducing cytotoxic agents [2,3]. DSBs recruit the damage sensor 53BP1 to the chromatin domain surrounding a DSB [7,8,9,10,11] where it co-localizes with γ-H2AX [7,9,12]. Radiation-induced DSBs can be addressed by microscopically visible DNA damage protein foci that display both γ-H2AX and 53BP1. DSB foci disappear by γ-H2AX dephosphorylation after a DSB is repaired [16]
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