Manabu Yasui1, Takayuki Fukuda2, Akiko Ukai3, Jiro Maniwa4, Tadashi Imamura5, Tsuneo Hashizume6, Haruna Yamamoto6, Kaori Shibuya6, Kazunori Narumi7, Yohei Fujiishi7, Emiko Okada7, Saori Fujishima8, Mika Yamamoto9, Naoko Otani9, Maki Nakamura2, Ryoichi Nishimura2, Maya Ueda10, Masayuki Mishima11, Kaori Matsuzaki11, Akira Takeiri11, Kenji Tanaka11, Yuki Okada12, Munehiro Nakagawa13, Shuichi Hamada2, Akihiko Kajikawa13, Hiroshi Honda14, Jun Adachi15, Kentaro Misaki16, Kumiko Ogawa17, Masamitsu Honma3. 1. Division of Genetics and Mutagenesis, National Institute of Health Sciences, 3-25-26 Tono-machi, Kawasaki-ku, Kawasaki, Kanagawa, 210-9501, Japan. m-yasui@nihs.go.jp. 2. Tokyo Laboratory, BoZo Research Center Inc., 1-3-11, Hanegi, Setagaya-ku, Tokyo, 156-0042, Japan. 3. Division of Genetics and Mutagenesis, National Institute of Health Sciences, 3-25-26 Tono-machi, Kawasaki-ku, Kawasaki, Kanagawa, 210-9501, Japan. 4. AstraZeneca KK, 3-1 Ofuka-cho, Kita-ku, Osaka, 530-0011, Japan. 5. Ina Research Inc., 2148-188 Nishiminowa, Ina-shi, Nagano, 399-4501, Japan. 6. Scientific Product Assessment Center, R&D Group, Japan Tobacco Inc., 6-2, Umegaoka, Aoba-ku, Yokohama, Kanagawa, 227-8512, Japan. 7. Yakult Central Institute, 5-11 Izumi, Kunitachi-shi, Tokyo, 186-8650, Japan. 8. Chemicals Evaluation and Research Institute, Japan, 3-822, Ishii-machi, Hita-shi, Oita, 877-0061, Japan. 9. Astellas Pharma Inc., 21, Miyukigaoka, Tsukuba-shi, Ibaraki, 305-8585, Japan. 10. Genotoxicology Laboratory, BioSafety Research Center Inc., 582-2 Shioshinden, Iwata-shi, Shizuoka, 437-1213, Japan. 11. Chugai Pharmaceutical Co., Ltd, 1-135, Komakado, Gotemba, Shizuoka, 412-8513, Japan. 12. Toxicology Research Department, Teijin Institute for Bio-medical Research, Teijin Pharma Limited, 4-3-2, Asahigaoka, Hino, Tokyo, 191-8512, Japan. 13. Nonclinical Research Center, LSI Medience Corporation, 14-1, Sunayama, Kamisu-shi, Ibaraki, 314-0255, Japan. 14. R&D Safety Science Research, Kao Corporation, Haga-Gun, Tochigi, Japan. 15. Laboratory of Proteomics for Drug Discovery, Center for Drug Design Research, National Institutes of Biomedical Innovation, Health and Nutrition, 7-6-8 Saito-Asagi, Ibarak, Osaka, 567-0085, Japan. 16. School of Nursing, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka, 422-8526, Japan. 17. Division of Pathology, National Institute of Health Sciences, 3-25-26 Tono-machi, Kawasaki-ku, Kawasaki, Kanagawa, 210-9501, Japan.
Abstract
BACKGROUND: 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). RESULTS: 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. CONCLUSIONS: 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.
BACKGROUND: Conflicting results between bacterial mutagenicity tests (the Ames test) and mammaliancarcinogenicity 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 humanlymphoblastoid 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). RESULTS: 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. CONCLUSIONS: 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.
Entities:
Keywords:
Ames test; Follow-up; Human lymphoblastoid TK6 cells; TK6 assay; Toxicoproteomics; Weight of evidence approach
Authors: Stefan Pfuhler; Ralph Pirow; Thomas R Downs; Andrea Haase; Nicola Hewitt; Andreas Luch; Marion Merkel; Claudia Petrick; André Said; Monika Schäfer-Korting; Kerstin Reisinger Journal: Mutagenesis Date: 2021-04-28 Impact factor: 3.000
Authors: Merle Marie Nicolai; Barbara Witt; Andrea Hartwig; Tanja Schwerdtle; Julia Bornhorst Journal: Arch Toxicol Date: 2021-08-30 Impact factor: 5.153