Eberlein Uta, Peper Michel, Fernández Maria, Lassmann Michael, Scherthan Harry
Department of Nuclear Medicine, University of Würzburg, Würzburg, Germany.
Bundeswehr Institute of Radiobiology affiliated to the University of Ulm, Munich, Germany.
PLoS One. 2015 Apr 8;10(4):e0123174. doi: 10.1371/journal.pone.0123174. eCollection 2015.
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.
电离辐射暴露诱导的DNA双链断裂（DSB）形成通过DSB生物标志物γ-H2AX和53BP1来指示。了解溶液中的放射性核素对全血样本进行内照射后体外DSB灶形成情况，将有助于我们深入了解分子放射治疗（MRT）后患者的剂量反应关系。因此，我们研究了体外辐射诱导的共定位γ-H2AX和53BP1灶的诱导情况，将其作为DSB的替代标志物，并将每个细胞获得的灶值与MRT中两种最常用放射性核素（I-131和Lu-177）对血液的体外吸收剂量相关联。这种方法得出了一条体外校准曲线。总体而言，分析了三名健康志愿者的55份血样。每次实验都制备了几个小瓶，其中含有全血和放射性溶液的混合物，这些放射性溶液是用不同浓度的等渗NaCl稀释的、具有已知活度的放射性核素。孵育并在37°C持续搅拌1小时后，通过密度离心回收白细胞。乙醇固定后，对其进行双色免疫荧光染色，并使用配备红/绿双带通滤光片的荧光显微镜确定共定位γ-H2AX和53BP1灶/细胞核的平均频率。通过校准的锗探测器测量并行确定每个血样的确切活度。通过蒙特卡罗模拟计算了两种同位素在1 ml血液中发生的每次核衰变对血液的吸收剂量率。我们样本中测得的血液剂量范围为6至95 mGy。在所研究的两种放射性核素中，均发现DSB标记灶/细胞核的数量与血液吸收剂量之间存在线性关系。仅存在微小的核素特异性个体内和个体间偏差。
