Follow-up genotoxicity assessment of Ames-positive/equivocal chemicals using the improved thymidine kinase gene mutation assay in DNA repair-deficient human TK6 cells
Follow-up genotoxicity assessment of Ames-positive/equivocal chemicals using the improved thymidine kinase gene mutation assay in DNA repair-deficient human TK6 cells
复制标题
在 DNA 修复缺陷的人 TK6 细胞中使用改进的胸苷激酶基因突变测定法对 Ames 阳性/模棱两可的化学物质进行后续遗传毒性评估
DOI：
10.1093/mutage/geab025
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发表时间：
2021
期刊：
影响因子：
2.7
通讯作者：
M. Yasui
中科院分区：
文献类型：
--
作者：
A. Sassa;T. Fukuda;A. Ukai;M. Nakamura;R. Sato;S. Fujiwara;K. Hirota;S. Takeda;K.I. Sugiyama;M. Honma;M. Yasui
遗传毒性试验在药物、农药和化学物质的安全性评价中起着重要作用。在各种遗传毒性试验指南中，体外遗传毒性试验组合包括细菌艾姆斯试验和哺乳动物细胞试验。几种化学品在细菌艾姆斯试验和哺乳动物细胞遗传毒性研究中显示出相互矛盾的结果，这可能源于不同细胞类型或种属之间DNA修复能力或代谢的差异。为了更好地理解不同检测系统之间结果冲突的机制含义，有必要开发对DNA损伤试剂具有更高特异性的体外遗传毒性检测方法。我们最近建立了一种改进的胸苷激酶（TK）基因突变检测（TK检测），即使用分别缺失碱基切除修复（BER）和核苷酸切除修复（NER）的核心因子XRCC 1和XPA（XRCC 1-/-/XPA-/-）的人淋巴母细胞TK 6细胞进行DNA切除修复系统缺陷检测。该DNA修复缺陷型TK 6细胞系有望根据DNA损伤特异性评价化学物质的遗传毒性潜力。我们重点研究了四种试剂，N-（1-萘基）乙二胺二盐酸盐（NEDA），对苯二胺（PPD），金胺和孔雀石绿色（MG）作为艾姆斯试验阳性化学品。在我们的试验中，使用XRCC 1-/-/XPA-/-细胞进行的评估显示，用NEDA、PPD和MG处理后，突变频率没有统计学显着增加，这表明这些化学物质对人类无遗传毒性。观察结果与后续体内研究一致。相反，在用金胺处理后，XRCC 1 −/−/XPA−/−细胞中的突变频率显著增加。结果表明，金胺是遗传毒性试剂，优先诱导DNA损伤解决BER和/或NER在哺乳动物中。综上所述，基于BER/NER缺陷细胞的遗传毒性试验将有助于阐明遗传毒性的机制，因此在化学物质的准确安全性评估中发挥关键作用。
Genotoxicity testing plays an important role in the safety assessment of pharmaceuticals, pesticides and chemical substances. Among the guidelines for various genotoxicity tests, thein vitrogenotoxicity test battery comprises the bacterial Ames test and mammalian cell assays. Several chemicals exhibit conflicting results for the bacterial Ames test and mammalian cell genotoxicity studies, which may stem from the differences in DNA repair capacity or metabolism, between different cell types or species. For better understanding the mechanistic implications regarding conflict outcomes between different assay systems, it is necessary to developin vitrogenotoxicity testing approaches with higher specificity towards DNA-damaging reagents. We have recently established an improved thymidine kinase (TK) gene mutation assay (TK assay) i.e. deficient in DNA excision repair system using human lymphoblastoid TK6 cells lackingXRCC1andXPA(XRCC1−/−/XPA−/−), the core factors of base excision repair (BER) and nucleotide excision repair (NER), respectively. This DNA repair-deficient TK6 cell line is expected to specifically evaluate the genotoxic potential of chemical substances based on the DNA damage. We focussed on four reagents,N-(1-naphthyl)ethylenediamine dihydrochloride (NEDA),p-phenylenediamine (PPD), auramine and malachite green (MG) as the Ames test-positive chemicals. In our assay, assessment usingXRCC1−/−/XPA−/−cells revealed no statistically significant increase in the mutant frequencies after treatment with NEDA, PPD and MG, suggesting the chemicals to be non-genotoxic in humans. The observations were consistent with that of the follow-upin vivostudies. In contrast, the mutant frequency was markedly increased inXRCC1−/−/XPA−/−cells after treatment with auramine. The results suggest that auramine is the genotoxic reagent that preferentially induces DNA damages resolved by BER and/or NER in mammals. Taken together, BER/NER-deficient cell-based genotoxicity testing will contribute to elucidate the mechanism of genotoxicity and therefore play a pivotal role in the accurate safety assessment of chemical substances.