Department of Radiation Genetics, Kyoto University, Graduate School of Medicine, Kyoto, Japan.
Division of Genetics and Mutagenesis, National Institute of Health Sciences, Kawasaki, Kanagawa, Japan.
Environ Mol Mutagen. 2020 Jul;61(6):602-610. doi: 10.1002/em.22371. Epub 2020 Apr 15.
The OECD guidelines define the bioassays of identifying mutagenic chemicals, including the thymidine kinase (TK) assay, which specifically detects the mutations that inactivate the TK gene in the human TK6 lymphoid line. However, the sensitivity of this assay is limited because it detects mutations occurring only in the TK gene but not any other genes. Moreover, the limited sensitivity of the conventional TK assay is caused by the usage of DNA repair-proficient wild-type cells, which are capable of accurately repairing DNA damage induced by chemicals. Mutagenic chemicals produce a variety of DNA lesions, including base lesions, sugar damage, crosslinks, and strand breaks. Base damage causes point mutations and is repaired by the base excision repair (BER) and nucleotide excision repair (NER) pathways. To increase the sensitivity of TK assay, we simultaneously disrupted two genes encoding XRCC1, an important BER factor, and XPA, which is essential for NER, generating XRCC1 /XPA cells from TK6 cells. We measured the mutation frequency induced by four typical mutagenic agents, methyl methane sulfonate (MMS), cis-diamminedichloro-platinum(II) (cisplatin, CDDP), mitomycin-C (MMC), and cyclophosphamide (CP) by the conventional TK assay using wild-type TK6 cells and also by the TK assay using XRCC1 /XPA cells. The usage of XRCC1 /XPA cells increased the sensitivity of detecting the mutagenicity by 8.6 times for MMC, 8.5 times for CDDP, and 2.6 times for MMS in comparison with the conventional TK assay. In conclusion, the usage of XRCC1 /XPA cells will significantly improve TK assay.
经合组织指南定义了识别诱变剂化学物质的生物测定法，包括胸苷激酶（TK）测定法，该测定法专门检测在人类 TK6 淋巴系中使 TK 基因失活的突变。然而，该测定法的灵敏度有限，因为它仅检测到发生在 TK 基因中的突变，而不是任何其他基因。此外，传统 TK 测定法的灵敏度有限是由于使用了具有 DNA 修复能力的野生型细胞，这些细胞能够准确修复化学物质诱导的 DNA 损伤。诱变剂化学物质会产生多种 DNA 损伤，包括碱基损伤、糖损伤、交联和链断裂。碱基损伤会导致点突变，并通过碱基切除修复（BER）和核苷酸切除修复（NER）途径进行修复。为了提高 TK 测定法的灵敏度，我们同时敲除了编码 XRCC1（BER 的重要因素）和 XPA（NER 所必需的）的两个基因，从而从 TK6 细胞中生成 XRCC1 / XPA 细胞。我们使用野生型 TK6 细胞的传统 TK 测定法和 XRCC1 / XPA 细胞的 TK 测定法，测量了四种典型诱变剂，即甲磺酸甲酯（MMS）、顺二氨二氯铂（II）（顺铂，CDDP）、丝裂霉素 C（MMC）和环磷酰胺（CP）诱导的突变频率。与传统 TK 测定法相比，使用 XRCC1 / XPA 细胞将 MMC 的致突变性检测灵敏度提高了 8.6 倍，将 CDDP 的致突变性检测灵敏度提高了 8.5 倍，将 MMS 的致突变性检测灵敏度提高了 2.6 倍。总之，使用 XRCC1 / XPA 细胞将显著提高 TK 测定法的灵敏度。
