Nishihara Kana, Huang Ruili, Zhao Jinghua, Shahane Sampada A, Witt Kristine L, Smith-Roe Stephanie L, Tice Raymond R, Takeda Shunichi, Xia Menghang
Radiation Genetics, Graduate School of Medicine, Kyoto University, Yoshidakonoe, Sakyo, Kyoto 606-8501, Japan, National Center for Advancing Translational Sciences, National Institutes of Health, 9800 Medical Center Drive, MSC: 3375 Bethesda, MD 20892, USA and.
National Center for Advancing Translational Sciences, National Institutes of Health, 9800 Medical Center Drive, MSC: 3375 Bethesda, MD 20892, USA and.
Mutagenesis. 2016 Jan;31(1):69-81. doi: 10.1093/mutage/gev055. Epub 2015 Aug 4.
DNA repair pathways play a critical role in maintaining cellular homeostasis by repairing DNA damage induced by endogenous processes and xenobiotics, including environmental chemicals. Induction of DNA damage may lead to genomic instability, disruption of cellular homeostasis and potentially tumours. Isogenic chicken DT40 B-lymphocyte cell lines deficient in DNA repair pathways can be used to identify genotoxic compounds and aid in characterising the nature of the induced DNA damage. As part of the US Tox21 program, we previously optimised several different DT40 isogenic clones on a high-throughput screening platform and confirmed the utility of this approach for detecting genotoxicants by measuring differential cytotoxicity in wild-type and DNA repair-deficient clones following chemical exposure. In the study reported here, we screened the Tox21 10K compound library against two isogenic DNA repair-deficient DT40 cell lines (KU70 (-/-) /RAD54 (-/-) and REV3 (-/-) ) and the wild-type cell line using a cell viability assay that measures intracellular adenosine triphosphate levels. KU70 and RAD54 are genes associated with DNA double-strand break repair processes, and REV3 is associated with translesion DNA synthesis pathways. Active compounds identified in the primary screening included many well-known genotoxicants (e.g. adriamycin, melphalan) and several compounds previously untested for genotoxicity. A subset of compounds was further evaluated by assessing their ability to induce micronuclei and phosphorylated H2AX. Using this comprehensive approach, three compounds with previously undefined genotoxicity-2-oxiranemethanamine, AD-67 and tetraphenylolethane glycidyl ether-were identified as genotoxic. These results demonstrate the utility of this approach for identifying and prioritising compounds that may damage DNA.
DNA修复途径通过修复内源性过程和包括环境化学物质在内的外源性物质诱导的DNA损伤，在维持细胞稳态中发挥关键作用。DNA损伤的诱导可能导致基因组不稳定、细胞稳态破坏以及潜在的肿瘤发生。缺乏DNA修复途径的同基因鸡DT40 B淋巴细胞系可用于鉴定遗传毒性化合物，并有助于表征诱导的DNA损伤的性质。作为美国Tox21计划的一部分，我们之前在高通量筛选平台上优化了几种不同的DT40同基因克隆，并通过测量化学暴露后野生型和DNA修复缺陷克隆中的差异细胞毒性，证实了该方法检测遗传毒性剂的实用性。在本文报道的研究中，我们使用一种测量细胞内三磷酸腺苷水平的细胞活力测定法，针对两种同基因DNA修复缺陷的DT40细胞系（KU70（-/-）/RAD54（-/-）和REV3（-/-））以及野生型细胞系，筛选了Tox21 10K化合物库。KU70和RAD54是与DNA双链断裂修复过程相关的基因，而REV3与跨损伤DNA合成途径相关。在初步筛选中鉴定出的活性化合物包括许多知名的遗传毒性剂（如阿霉素、美法仑）以及几种之前未进行遗传毒性测试的化合物。通过评估它们诱导微核和磷酸化H2AX的能力，对一部分化合物进行了进一步评估。使用这种综合方法，鉴定出三种之前遗传毒性未明确的化合物——2-氧杂环丙烷甲胺、AD-67和四苯基乙烷缩水甘油醚——具有遗传毒性。这些结果证明了该方法在鉴定和优先排序可能损害DNA的化合物方面的实用性。
