Hu V W, Heikka D S, Dieffenbach P B, Ha L
Department of Biochemistry and Molecular Biology, The George Washington University, School of Medicine and Health Sciences, Washington, DC 20037, USA.
FASEB J. 2001 Jul;15(9):1562-8. doi: 10.1096/fj.01-0102com.
Despite the general assumption that widely used radiolabeled metabolites such as [(35)S]methionine and (3)H-thymidine do not adversely affect or perturb cell function, we and others have shown that such low-energy beta-emitters can cause cell cycle arrest and apoptosis of proliferating cells. The goal of the present study was to elucidate the targets and mechanisms of [(35)S]methionine-induced cellular toxicity. Comet analyses (single-cell electrophoresis) demonstrated dose-dependent DNA fragmentation in rabbit smooth muscle cells within a time frame (1-4 h) well within that of most radiolabeling protocols, whereas fluorescence analyses using a peroxide/hydroperoxide-sensitive dye revealed production of reactive oxygen species (ROS). Although ROS generation was inhibitable by antioxidants, DNA fragmentation was not inhibited and was in fact observed even under hypoxic conditions, suggesting that beta-radiation-induced DNA damage can occur independently of ROS formation. Studies with p53(+/+) and p53(-/-) human colorectal carcinoma cells further demonstrated the dissociation of early DNA damage from ROS formation in that both cell types exhibited DNA fragmentation in response to radiolabeling whereas only the p53(+/+) cells exhibited significant increases in ROS formation, which occurred well after significant DNA damage was observed. These findings demonstrate that metabolically incorporated low-energy beta-emitters such as [(35)S]methionine and (3)H-thymidine can induce DNA damage, thereby initiating cellular responses leading to cell cycle arrest or apoptosis. The results of this study require a reevaluation using low-energy beta-emitters to follow not only experimental protocols in vivo processes, but also acceptable exposure levels of these genotoxic compounds in the workplace and environment.
尽管人们普遍认为广泛使用的放射性标记代谢物，如[（35）S]蛋氨酸和3H-胸腺嘧啶核苷不会对细胞功能产生不利影响或干扰，但我们和其他人已经表明，这种低能β发射体可导致增殖细胞的细胞周期停滞和凋亡。本研究的目的是阐明[（35）S]蛋氨酸诱导的细胞毒性的靶点和机制。彗星分析（单细胞电泳）显示，在大多数放射性标记方案的时间范围内（1-4小时），兔平滑肌细胞中存在剂量依赖性DNA片段化，而使用对过氧化物/过氧化氢敏感的染料进行的荧光分析显示产生活性氧（ROS）。尽管抗氧化剂可抑制ROS的产生，但DNA片段化并未受到抑制，事实上，即使在缺氧条件下也能观察到DNA片段化，这表明β辐射诱导的DNA损伤可独立于ROS形成而发生。对p53（+/+）和p53（-/-）人结肠癌细胞的研究进一步证明了早期DNA损伤与ROS形成的分离，因为两种细胞类型在接受放射性标记后均表现出DNA片段化，而只有p53（+/+）细胞表现出ROS形成的显著增加，这发生在观察到显著DNA损伤之后。这些发现表明，代谢掺入的低能β发射体，如[（35）S]蛋氨酸和3H-胸腺嘧啶核苷，可诱导DNA损伤，从而引发导致细胞周期停滞或凋亡的细胞反应。本研究结果要求重新评估使用低能β发射体的情况，不仅要评估体内过程的实验方案，还要评估这些遗传毒性化合物在工作场所和环境中的可接受暴露水平。
