Abstract
BackgroundThe 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 γH2AX, 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 γH2AX foci in human lymphoblastoid TK6 cells.ResultsTK6 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 γH2AX 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 γH2AX foci and MNi, while the aneugens induced only MNi, not γH2AX 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.ConclusionsA HC imaging assay to detect γH2AX foci and MNi in TK6 cells was established, and the assay provided information on the aneugenic/clastogenic mode of action.
Highlights
The in vitro micronucleus (MN) test is an important component of a genotoxicity test battery [1]
Preparation of plate specimens and imaging analysis Fixation of TK6 cells on the bottom of 96-well plates was successfully achieved by the protocol established in this study (Fig. 1)
When conditions were without S9 mix, both MNi and γH2AX foci were analyzed simultaneously using the common image sources
Summary
The in vitro micronucleus (MN) test is an important component of a genotoxicity test battery [1]. Manual scoring by microscope is a reliable and standard method used so far to detect micronucleated (MNed) cells, the method is laborious and time-consuming. Revealing the clastogenic or aneugenic mode of action (MoA) is important, especially in pharmaceutical development, which needs to define a threshold of genotoxicity [2]. It is especially important to elucidate an aneugenic MoA at an early stage of pharmaceutical development, because this can avoid eliminating promising candidates on which sufficient safety margins could be set. The in vitro micronucleus (MN) test is an important component of a genotoxicity test battery that evaluates chemicals. A high-throughput assay system for detecting MN cells automatically has long been desired in the fields of pharmaceutical development or environmental risk monitoring. We aimed to establish a high-content (HC) imaging assay that automatically detects micronuclei (MNi) and simultaneously measures γH2AX foci in human lymphoblastoid TK6 cells
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