Gault Nathalie, Rigaud Odile, Poncy Jean-Luc, Lefaix Jean-Louis
Commissariat à l'Energie Atomique, Direction des Sciences du Vivant, Département de Radiobiologie-Radiopathologie, 92265 Fontenay aux Roses, France.
Radiat Res. 2007 May;167(5):551-62. doi: 10.1667/RR0684.1.
Irradiation of individual cell nuclei with charged-particle microbeams requires accurate identification and localization of cells using Hoechst staining and UV illumination before computer-monitored localization of each cell. Using Fourier-transform infrared microspectroscopy (FT-IRM), we investigated whether the experimental conditions used for cell recognition induce cellular changes prior to irradiation and compared biochemical changes and DNA damage after targeted and nontargeted irradiation with alpha particles delivered by macro- or microbeams, using gamma radiation as a reference. Molecular damage in single HaCaT cells was studied by means of FT-IRM and comet assay (Gault et al., Int. J. Radiat. Biol. 81, 767-779, 2005). Hoechst 33342-stained HaCaT cells were exposed to single doses of 2 Gy (239)Pu alpha particles from a broad-beam irradiator, five impacted alpha particles from a microbeam irradiator, or 6 Gy gamma rays from (137)Cs, each of which resulted in about 5% clonogenic survival. FT-IRM of control cells indicated that Hoechst binding to nuclear DNA induced subtle changes in DNA conformation, and its excitation under UV illumination induced a dramatic shift of the DNA conformation from A to B as well as major DNA damage as measured by the comet assay. Comparison of the FT-IRM spectra of cells exposed to gamma rays or alpha particles specifically targeted to the nucleus, alpha particles from a broad-beam irradiator revealed spectral changes corresponding to all changes in constitutive bases in nucleic acids, suggesting oxidative damage in these bases, as well as structural damage in the deoxyribose-phosphate backbone of DNA and the osidic structure of nucleic acids. Concomitantly, spectral changes specific to protein suggested structural modifications. Striking differences in IR spectra between targeted microbeam- and nontargeted macrobeam-irradiated cells indicated greater residual unrepaired or misrepaired damage after microbeam irradiation. This was confirmed by the comet assay data. These results show that FT-IRM, together with the comet assay, is useful for assessing direct radiation-induced damage to nucleic acids and proteins in single cells and for investigating the effects of radiation quality. Significantly, FT-IRM revealed that Hoechst 33342 binding to DNA and exposure to UV light induce a dramatic change in DNA conformation as well as DNA damage. These findings suggest that fluorochrome staining should be avoided in studies of ionizing radiation-induced bystander effects based on charged-particle microbeam irradiation. An alternative cell nucleus recognition system that avoids nuclear matrix damage and its possible contribution to propagation of biological effects from irradiated cells to neighboring nontargeted cells needs to be developed.
使用带电粒子微束对单个细胞核进行照射，在通过计算机监测对每个细胞进行定位之前，需要利用Hoechst染色和紫外线照明对细胞进行准确识别和定位。我们使用傅里叶变换红外光谱（FT-IRM）研究了用于细胞识别的实验条件是否会在照射前引起细胞变化，并以γ射线为参照，比较了用宏观或微观束流递送的α粒子进行靶向和非靶向照射后的生化变化及DNA损伤。通过FT-IRM和彗星试验研究了单个HaCaT细胞中的分子损伤（Gault等人，《国际辐射生物学杂志》81卷，767 - 779页，2005年）。用Hoechst 33342染色的HaCaT细胞分别接受来自宽束辐照器的2 Gy （239）Puα粒子单剂量照射、来自微束辐照器的5个撞击α粒子照射或来自（137）Cs的6 Gyγ射线照射，每种照射均导致约5%的克隆形成存活率。对照细胞FT-IRM结果表明，Hoechst与核DNA结合会引起DNA构象的细微变化，其在紫外线照射下的激发会导致DNA构象从A向B发生显著转变，同时通过彗星试验测量发现有严重的DNA损伤。对接受γ射线或靶向细胞核的α粒子照射的细胞的FT-IRM光谱进行比较，来自宽束辐照器的α粒子照射的细胞显示出与核酸组成碱基的所有变化相对应的光谱变化，表明这些碱基存在氧化损伤，以及DNA的脱氧核糖 - 磷酸骨架和核酸糖苷结构的结构损伤。同时，蛋白质特有的光谱变化表明存在结构修饰。靶向微束照射和非靶向宏观束照射的细胞的红外光谱存在显著差异，表明微束照射后有更多未修复或错配修复的残留损伤。彗星试验数据证实了这一点。这些结果表明，FT-IRM与彗星试验一起，可用于评估辐射对单个细胞中核酸和蛋白质的直接损伤，并用于研究辐射质量的影响。重要的是，FT-IRM显示Hoechst 33342与DNA结合以及暴露于紫外线下会导致DNA构象发生显著变化以及DNA损伤。这些发现表明，在基于带电粒子微束照射的电离辐射旁观者效应研究中应避免使用荧光染料染色。需要开发一种替代的细胞核识别系统，以避免核基质损伤及其可能对生物效应从受照射细胞传播到相邻非靶向细胞的贡献。
