由内在和/或外在活性氧（ROS）介导的细胞氧化应激与疾病发病机制相关。 DNA 氧化损伤自然可以被线粒体 DNA (mtDNA) 取代，导致碱基损伤/链断裂形成、拷贝数变化和突变。在这项研究中，我们设计了一种使用超螺旋敏感定量 PCR (ss-qPCR) 来灵敏定量急性 mtDNA 损伤、修复和拷贝数变化的单一测试，并检查了口腔癌细胞中氧化应激相关的 mtDNA 损伤反应是如何发生的。我们观察到，外源过氧化氢 (H2O2) 会诱导动态 mtDNA 损伤反应，如早期结构 DNA 损伤所反映的那样，如果损伤没有超过特定阈值，则随后进行 DNA 修复。然而，高氧化应激水平会诱导持续的 mtDNA 损伤，并导致晚期反应中 mtDNA 拷贝数减少 5-30 倍。这种急剧的消耗与显着的生长停滞和细胞凋亡相关，表明持续的功能后果。此外，与正常细胞相比，口腔癌细胞对氧化损伤的反应不同，并且不同的ROS种类在应激条件下引发不同的生物学后果。总之，我们开发了一种灵敏检测 mtDNA 损伤和拷贝数变化的新方法，利用外源性 H2O2 诱导与应激癌细胞功能变化相关的动态 mtDNA 损伤反应。最后，我们的方法可以帮助表征癌症和其他人类疾病中的氧化 DNA 损伤。版权所有：© 2024 Tan 等人。这是一篇根据知识共享署名许可条款分发的开放获取文章，允许在任何媒体上不受限制地使用、分发和复制，前提是注明原始作者和来源。

Cellular oxidative stress mediated by intrinsic and/or extrinsic reactive oxygen species (ROS) is associated with disease pathogenesis. Oxidative DNA damage can naturally be substituted by mitochondrial DNA (mtDNA), leading to base lesion/strand break formation, copy number changes, and mutations. In this study, we devised a single test for the sensitive quantification of acute mtDNA damage, repair, and copy number changes using supercoiling-sensitive quantitative PCR (ss-qPCR) and examined how oxidative stress-related mtDNA damage responses occur in oral cancer cells. We observed that exogenous hydrogen peroxide (H2O2) induced dynamic mtDNA damage responses, as reflected by early structural DNA damage, followed by DNA repair if damage did not exceed a particular threshold. However, high oxidative stress levels induced persistent mtDNA damage and caused a 5-30-fold depletion in mtDNA copy numbers over late responses. This dramatic depletion was associated with significant growth arrest and apoptosis, suggesting persistent functional consequences. Moreover, oral cancer cells responded differentially to oxidative injury when compared with normal cells, and different ROS species triggered different biological consequences under stress conditions. In conclusion, we developed a new method for the sensitive detection of mtDNA damage and copy number changes, with exogenous H2O2 inducing dynamic mtDNA damage responses associated with functional changes in stressed cancer cells. Finally, our method can help characterize oxidative DNA damage in cancer and other human diseases.Copyright: © 2024 Tan et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.