细胞暴露于高能量传递密度较高且离散分布的低线性能量传递(LET)辐射(各占50%剂量)时，其反应比单独接受相同剂量的辐射更为强烈。本研究使用高分辨透射电子显微镜(TEM)评估了DNA双链断裂在细胞核内的位置与染色质结构之间的关系，采用乳腺癌MDA-MB-231细胞在5 Gy剂量和30分钟后进行了观察。此外，通过单次(1×1.5 Gy)与多次分次给药(5×0.3 Gy)评估了对高和/或低LET辐射的反应。通过TEM分析，我们发现接受阿尔法辐射后的伽马辐射(称为混合束)引起的γH2AX纳米珠总数最高，其次是阿尔法辐射，然后是伽马辐射。与辐射引起的γH2AX灶相比，混合辐射所引起的γH2AX灶倾向于被开放染色质(较浅的TEM区域)所环绕，然而具有最多珠子数即代表复杂损伤的较大灶仍保留在畴状区域内。通过免疫荧光分析，我们发现混合辐射处理细胞中γH2AX大灶面积也较大。每日给予分次混合束辐射在MDA-MB-231和骨肉瘤U2OS细胞中比其他辐射质量以及急性暴露引起了最强的细胞存活率和克隆形成抑制效应。这可能部分原因是每一次分次给药产生了重复性低LET氧化性DNA损伤，同时在最后一次混合束分次给药后2小时观察到的heterochromatin标记物H3K9me3水平较低，表明高LET辐射后染色质的重新压缩受到了延迟。综上所述，对复杂DNA损伤的早期差异反应可能导致分次辐射引起更强的细胞杀伤作用，这表明了联合高能量传递和低能量传递辐射的治疗潜力。© 2023 The Authors. Published by Elsevier B.V. All rights reserved.

Cells exposed to densely ionising high and scattered low linear energy transfer (LET) radiation (50 % dose of each) react more strongly than to the same dose of each separately. The relationship between DNA double strand break location inside the nucleus and chromatin structure was evaluated, using high-resolution transmission electron microscopy (TEM) in breast cancer MDA-MB-231 cells at 30 min post 5 Gy. Additionally, response to high and/or low LET radiation was assessed using single (1 ×1.5 Gy) versus fractionated dose delivery (5 ×0.3 Gy). By TEM analysis, the highest total number of γH2AX nanobeads were found in cells irradiated with alpha radiation just prior to gamma radiation (called mixed beam), followed by alpha, then gamma radiation. γH2AX foci induced by mixed beam radiation tended to be surrounded by open chromatin (lighter TEM regions), yet foci containing the highest number of beads, i.e. larger foci representing complex damage, remained in the heterochromatic areas. The γH2AX large focus area was also greater in mixed beam-treated cells when analysed by immunofluorescence. Fractionated mixed beams given daily induced the strongest reduction in cell viability and colony formation in MDA-MB-231 and osteosarcoma U2OS cells compared to the other radiation qualities, as well as versus acute exposure. This may partially be explained by recurring low LET oxidative DNA damage by every fraction together with a delay in recompaction of chromatin after high LET, demonstrated by low levels of heterochromatin marker H3K9me3 at 2 h after the last mixed beam fraction in MDA-MB-231. In conclusion, early differences in response to complex DNA damage may lead to a stronger cell kill induced by fractionated exposure, which suggest a therapeutic potential of combined high and low LET irradiation.Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.