Yasui Manabu, Fukuda Takayuki, Ukai Akiko, Maniwa Jiro, Imamura Tadashi, Hashizume Tsuneo, Yamamoto Haruna, Shibuya Kaori, Narumi Kazunori, Fujiishi Yohei, Okada Emiko, Fujishima Saori, Yamamoto Mika, Otani Naoko, Nakamura Maki, Nishimura Ryoichi, Ueda Maya, Mishima Masayuki, Matsuzaki Kaori, Takeiri Akira, Tanaka Kenji, Okada Yuki, Nakagawa Munehiro, Hamada Shuichi, Kajikawa Akihiko, Honda Hiroshi, Adachi Jun, Misaki Kentaro, Ogawa Kumiko, Honma Masamitsu
Division of Genetics and Mutagenesis, National Institute of Health Sciences, 3-25-26 Tono-machi, Kawasaki-ku, Kawasaki, Kanagawa, 210-9501, Japan.
Tokyo Laboratory, BoZo Research Center Inc., 1-3-11, Hanegi, Setagaya-ku, Tokyo, 156-0042, Japan.
Genes Environ. 2021 Mar 6;43(1):7. doi: 10.1186/s41021-021-00179-1.
Conflicting results between bacterial mutagenicity tests (the Ames test) and mammalian carcinogenicity tests might be due to species differences in metabolism, genome structure, and DNA repair systems. Mutagenicity assays using human cells are thought to be an advantage as follow-up studies for positive results in Ames tests. In this collaborative study, a thymidine kinase gene mutation study (TK6 assay) using human lymphoblastoid TK6 cells, established in OECD TG490, was used to examine 10 chemicals that have conflicting results in mutagenicity studies (a positive Ames test and a negative result in rodent carcinogenicity studies).
Two of 10 test substances were negative in the overall judgment (20% effective as a follow-up test). Three of these eight positive substances were negative after the short-term treatment and positive after the 24 h treatment, despite identical treatment conditions without S9. A toxicoproteomic analysis of TK6 cells treated with 4-nitroanthranilic acid was thus used to aid the interpretation of the test results. This analysis using differentially expressed proteins after the 24 h treatment indicated that in vitro specific oxidative stress is involved in false positive response in the TK6 assay.
The usefulness of the TK6 assay, by current methods that have not been combined with new technologies such as proteomics, was found to be limited as a follow-up test, although it still may help to reduce some false positive results (20%) in Ames tests. Thus, the combination analysis with toxicoproteomics may be useful for interpreting false positive results raised by 24 h specific reactions in the assay, resulting in the more reduction (> 20%) of false positives in Ames test.
细菌致突变性试验（艾姆斯试验）与哺乳动物致癌性试验结果相互矛盾，可能是由于代谢、基因组结构和DNA修复系统存在物种差异。使用人类细胞的致突变性检测被认为是对艾姆斯试验阳性结果进行后续研究的一个优势。在这项合作研究中，采用经合组织TG490中建立的人淋巴母细胞TK6细胞进行胸苷激酶基因突变研究（TK6检测），以检测10种在致突变性研究中结果相互矛盾的化学物质（艾姆斯试验呈阳性而啮齿动物致癌性试验呈阴性）。
10种受试物质中有2种在总体判断中呈阴性（作为后续检测的有效率为20%）。尽管在无S9的相同处理条件下，这8种阳性物质中有3种在短期处理后呈阴性，而在24小时处理后呈阳性。因此，对用4-硝基邻氨基苯甲酸处理的TK6细胞进行了毒理蛋白质组学分析，以辅助解释检测结果。对24小时处理后差异表达蛋白质的分析表明，体外特异性氧化应激参与了TK6检测中的假阳性反应。
虽然TK6检测仍可能有助于减少艾姆斯试验中的一些假阳性结果（20%），但通过目前尚未与蛋白质组学等新技术相结合的方法，发现其作为后续检测的作用有限。因此，与毒理蛋白质组学的联合分析可能有助于解释该检测中24小时特异性反应产生的假阳性结果，从而进一步减少艾姆斯试验中的假阳性（>20%）。
