a Laboratory of Radiation Biology, Department of Applied Veterinary Sciences, Faculty of Veterinary Medicine , Hokkaido University , Sapporo , Japan.
b Laboratory of Toxicology, Department of Environmental Veterinary Science, Faculty of Veterinary Medicine , Hokkaido University , Sapporo , Japan.
Free Radic Res. 2018 Jun;52(6):648-660. doi: 10.1080/10715762.2018.1460472. Epub 2018 Apr 19.
To evaluate the metabolic responses in tumour cells exposed to ionizing radiation, oxygen consumption rate (OCR), cellular lipid peroxidation, cellular energy status (intracellular nucleotide pool and ATP production), and mitochondrial reactive oxygen species (ROS), semiquinone (SQ), and iron-sulphur (Fe-S) cluster levels were evaluated in human cervical carcinoma HeLa cells at 12 and 24 h after X-irradiation. LC/MS/MS analysis showed that levels of 8-iso PGF and 5-iPF-VI, lipid peroxidation products of membrane arachidonic acids, were not altered significantly in X-irradiated cells, although mitochondrial ROS levels and OCR significantly increased in the cells at 24 h after irradiation. LC/UV analysis revealed that intracellular AMP, ADP, and ATP levels increased significantly after X-irradiation, but adenylate energy charge (adenylate energy charge (AEC) = [ATP + 0.5 × ADP]/[ATP + ADP + AMP]) remained unchanged after X-irradiation. In low-temperature electron spin resonance (ESR) spectra of HeLa cells, the presence of mitochondrial SQ at g = 2.004 and Fe-S cluster at g = 1.941 was observed and X-irradiation enhanced the signal intensity of SQ but not of the Fe-S cluster. Furthermore, this radiation-induced increase in SQ signal intensity disappeared on treatment with rotenone, which inhibits electron transfer from Fe-S cluster to SQ in complex I. From these results, it was suggested that an increase in OCR and imbalance in SQ and Fe-S cluster levels, which play a critical role in the mitochondrial electron transport chain (ETC), occur after X-irradiation, resulting in an increase in ATP production and ROS leakage from the activated mitochondrial ETC.
为了评估肿瘤细胞暴露在电离辐射下的代谢反应，我们在 X 射线照射后 12 和 24 小时评估了人宫颈癌细胞系 HeLa 中的耗氧率（OCR）、细胞脂质过氧化、细胞能量状态（细胞内核苷酸池和 ATP 生成）以及线粒体活性氧（ROS）、半醌（SQ）和铁硫（Fe-S）簇水平。LC/MS/MS 分析显示，X 射线照射的细胞中膜花生四烯酸的脂质过氧化产物 8-isoPGF 和 5-iPF-VI 的水平没有明显改变，尽管线粒体 ROS 水平和 OCR 在照射后 24 小时的细胞中显著增加。LC/UV 分析显示，X 射线照射后细胞内 AMP、ADP 和 ATP 水平显著增加，但 X 射线照射后腺苷酸能荷（AEC= [ATP + 0.5 × ADP] / [ATP + ADP + AMP]）保持不变。在 HeLa 细胞的低温电子自旋共振（ESR）谱中，观察到线粒体 SQ 在 g=2.004 和 Fe-S 簇在 g=1.941 的存在，X 射线照射增强了 SQ 的信号强度，但不增强 Fe-S 簇的信号强度。此外，这种辐射诱导的 SQ 信号强度增加在鱼藤酮处理后消失，鱼藤酮抑制了复合物 I 中从 Fe-S 簇到 SQ 的电子转移。从这些结果表明，X 射线照射后 OCR 增加以及 SQ 和 Fe-S 簇水平失衡，这在线粒体电子传递链（ETC）中起着关键作用，导致 ATP 生成增加和激活的线粒体 ETC 中 ROS 漏出。
