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Enhancing the sensitivity of the thymidine kinase assay by using DNA repair‐deficient human TK6 cells
Author(s) -
Ibrahim Mahmoud Abdelghany,
Yasui Manabu,
Saha Liton Kumar,
Sasanuma Hiroyuki,
Honma Masamitsu,
Takeda Shunichi
Publication year - 2020
Publication title -
environmental and molecular mutagenesis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1
H-Index - 87
eISSN - 1098-2280
pISSN - 0893-6692
DOI - 10.1002/em.22371
Subject(s) - xrcc1 , microbiology and biotechnology , thymidine kinase , dna damage , biology , base excision repair , dna repair , nucleotide excision repair , methane sulfonate , mutagenesis , xeroderma pigmentosum , thymidine , dna , mutation , mutant , gene , biochemistry , genetics , virus , herpes simplex virus , genotype , single nucleotide polymorphism
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.