학술논문
High-content imaging analyses of [gamma]H2AX-foci and micronuclei in TK6 cells elucidated genotoxicity of chemicals and their clastogenic/aneugenic mode of action
Document Type
Report
Author
Source
Genes and Environment. February 5, 2019, Vol. 41 Issue 1
Subject
Language
English
ISSN
1880-7046
Abstract
Author(s): Akira Takeiri[sup.1] , Kaori Matsuzaki[sup.1] , Shigeki Motoyama[sup.1] , Mariko Yano[sup.1] , Asako Harada[sup.1] , Chiaki Katoh[sup.1] , Kenji Tanaka[sup.1] and Masayuki Mishima[sup.1] Introduction The in vitro micronucleus (MN) [...]
Background The in vitro micronucleus (MN) test is an important component of a genotoxicity test battery that evaluates chemicals. Although the standard method of manually scoring micronucleated (MNed) cells by microscope is a reliable and standard method, it is laborious and time-consuming. A high-throughput assay system for detecting MN cells automatically has long been desired in the fields of pharmaceutical development or environmental risk monitoring. Although the MN test per se cannot clarify whether the mode of MN induction is aneugenic or clastogenic, this clarification may well be made possible by combining the MN test with an evaluation of [gamma]H2AX, a sensitive marker of DNA double strand breaks (DSB). In the present study, we aimed to establish a high-content (HC) imaging assay that automatically detects micronuclei (MNi) and simultaneously measures [gamma]H2AX foci in human lymphoblastoid TK6 cells. Results TK6 cells were fixed on the bottom of each well in 96-well plates hypotonically, which spreads the cells thinly to detach MNi from the primary nuclei. Then, the number of MNi and immunocytochemically-stained [gamma]H2AX foci were measured using an imaging analyzer. The system correctly judged 4 non-genotoxins and 13 genotoxins, which included 9 clastogens and 4 aneugens representing various genotoxic mechanisms, such as DNA alkylation, cross-linking, topoisomerase inhibition, and microtubule disruption. Furthermore, all the clastogens induced both [gamma]H2AX foci and MNi, while the aneugens induced only MNi, not [gamma]H2AX foci; therefore, the HC imaging assay clearly discriminated the aneugens from the clastogens. Additionally, the test system could feasibly analyze cell cycle, to add information about a chemical's mode of action. Conclusions A HC imaging assay to detect [gamma]H2AX foci and MNi in TK6 cells was established, and the assay provided information on the aneugenic/clastogenic mode of action. Keywords: High content, Genotoxicity, Screening, [gamma]H2AX, Micronucleus, TK6
Background The in vitro micronucleus (MN) test is an important component of a genotoxicity test battery that evaluates chemicals. Although the standard method of manually scoring micronucleated (MNed) cells by microscope is a reliable and standard method, it is laborious and time-consuming. A high-throughput assay system for detecting MN cells automatically has long been desired in the fields of pharmaceutical development or environmental risk monitoring. Although the MN test per se cannot clarify whether the mode of MN induction is aneugenic or clastogenic, this clarification may well be made possible by combining the MN test with an evaluation of [gamma]H2AX, a sensitive marker of DNA double strand breaks (DSB). In the present study, we aimed to establish a high-content (HC) imaging assay that automatically detects micronuclei (MNi) and simultaneously measures [gamma]H2AX foci in human lymphoblastoid TK6 cells. Results TK6 cells were fixed on the bottom of each well in 96-well plates hypotonically, which spreads the cells thinly to detach MNi from the primary nuclei. Then, the number of MNi and immunocytochemically-stained [gamma]H2AX foci were measured using an imaging analyzer. The system correctly judged 4 non-genotoxins and 13 genotoxins, which included 9 clastogens and 4 aneugens representing various genotoxic mechanisms, such as DNA alkylation, cross-linking, topoisomerase inhibition, and microtubule disruption. Furthermore, all the clastogens induced both [gamma]H2AX foci and MNi, while the aneugens induced only MNi, not [gamma]H2AX foci; therefore, the HC imaging assay clearly discriminated the aneugens from the clastogens. Additionally, the test system could feasibly analyze cell cycle, to add information about a chemical's mode of action. Conclusions A HC imaging assay to detect [gamma]H2AX foci and MNi in TK6 cells was established, and the assay provided information on the aneugenic/clastogenic mode of action. Keywords: High content, Genotoxicity, Screening, [gamma]H2AX, Micronucleus, TK6