Differences in quantification of DNA double-strand breaks assessed by 53BP1/γH2AX focus formation assays and the comet assay in mammalian cells treated with irradiation and N-acetyl-L-cysteine.
Differences in quantification of DNA double-strand breaks assessed by 53BP1/γH2AX focus formation assays and the comet assay in mammalian cells treated with irradiation and N-acetyl-L-cysteine.
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DOI：
10.1093/jrr/rrw001
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发表时间：
2016-06
影响因子：
2
通讯作者：
Nagayama Y
中科院分区：
文献类型：
--
作者：
Kurashige T;Shimamura M;Nagayama Y
电离辐射（IR）对基因组DNA的生物学效应被认为是直接或间接的;后者是通过IR诱导自由基和活性氧（ROS）介导的。本研究旨在评价N-乙酰-L-半胱氨酸（NAC），一种众所周知的ROS清除抗氧化剂，对哺乳动物细胞的遗传毒性，细胞毒性和ROS产生的IR诱导的影响，并旨在澄清以前的出版物中相互矛盾的数据。虽然我们清楚地证明了NAC对IR诱导的遗传毒性和细胞毒性的有益作用（使用微核试验和细胞活力/克隆形成试验测定），但NAC对DNA双链断裂（DSB）形成的影响的数据在不同的试验中不一致。具体而言，NAC对IR诱导的DSB的缓解可通过中性彗星试验检测到，但不能通过γ H2 AX或53 BP 1病灶试验检测到。NAC是谷胱甘肽前体，转化为谷胱甘肽后发挥作用，推测其具有自身的生物活性。假设焦点试验反映了对DSB的生物学反应（检测和修复），而彗星试验反映了基因组DNA的物理状态，我们的结果表明，彗星试验可以很容易地检测NAC对DSB形成的抗氧化作用。然而，NAC的生物学效应可能会影响病灶检测DSB修复。我们的数据表明，在研究辐射防护化合物的潜在候选物时，应仔细使用多个参数来分析DNA损伤。
The biological effect of ionizing radiation (IR) on genomic DNA is thought to be either direct or indirect; the latter is mediated by IR induction of free radicals and reactive oxygen species (ROS). This study was designed to evaluate the effect of N-acetyl-L-cysteine (NAC), a well-known ROS-scavenging antioxidant, on IR induction of genotoxicity, cytotoxicity and ROS production in mammalian cells, and aimed to clarify the conflicting data in previous publications. Although we clearly demonstrate the beneficial effect of NAC on IR-induced genotoxicity and cytotoxicity (determined using the micronucleus assay and cell viability/clonogenic assays), the data on NAC's effect on DNA double-strand break (DSB) formation were inconsistent in different assays. Specifically, mitigation of IR-induced DSBs by NAC was readily detected by the neutral comet assay, but not by the γH2AX or 53BP1 focus assays. NAC is a glutathione precursor and exerts its effect after conversion to glutathione, and presumably it has its own biological activity. Assuming that the focus assay reflects the biological responses to DSBs (detection and repair), while the comet assay reflects the physical status of genomic DNA, our results indicate that the comet assay could readily detect the antioxidant effect of NAC on DSB formation. However, NAC's biological effect might affect the detection of DSB repair by the focus assays. Our data illustrate that multiple parameters should be carefully used to analyze DNA damage when studying potential candidates for radioprotective compounds.