Chromosomal aberrations  clastogens vs aneugens

Mishima, Masayuki

doi:10.2741/s468

Cited by 31 publications

(12 citation statements)

References 115 publications

(136 reference statements)

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“…The so‐called linear‐no‐threshold model was generally applied to genotoxicological risk assessment. Numerous studies have shown nonlinear genotoxic mechanisms predominantly associated with aneuploidy‐inducing agents (Bolt et al, ; Crump, ; Elhajouji et al, ; ICH, ; Bevan and Harrison, ; Mishima, ), but also with clastogens acting as indirect inducers of DNA double‐strand breaks (Lynch et al, ; Bryce et al, ; Johnson et al, ). Although the primary goal of this work was not the demonstration of nonlinear dose–response metrics, the concentrations used for ETO allowed us to identify a no‐observed genotoxic effect level at 0.5 μmol/L.…”

Section: Discussionmentioning

confidence: 99%

“…In general, genotoxic compounds like clastogens and aneugens can induce micronuclei (MN). MN are small, extra‐nuclear chromatin bodies surrounded by a nuclear envelope, which are not incorporated into the nucleus of a daughter cell after nuclear division (Fenech and Morley, ; Fenech, ; Vanhauwaert et al, ; Mishima, ). MN are indicative of chromosome or genome mutations and are commonly observed in cancer development (Kirsch‐Volders et al, ; Bolognesi et al, ; Morita et al, ).…”

Section: Introductionmentioning

confidence: 99%

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Differentiation of Aneugens and Clastogens in the In Vitro Micronucleus Test by Kinetochore Scoring Using Automated Image Analysis

Wilde

Queisser

Holz³

et al. 2018

Environ and Mol Mutagen

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The in vitro micronucleus test according to OECD Test Guideline 487 (TG 487) is widely used to investigate the genotoxic potential of drugs. Besides the identification of in vitro genotoxicants, the assay can be complemented with kinetochore staining for the differentiation between clastogens and aneugens. This differentiation constitutes a major contribution to risk assessment as especially aneugens show a threshold response. Thus, a novel method for automated MN plus kinetochore (k+) scoring by image analysis was developed based on the OECD TG 487. Compound‐induced increases in MN frequency can be detected using the cytokinesis‐block (cytochalasin B) method in V79 cells after 24 h in a 96‐well format. Nuclei, MN, and kinetochores were labeled with nuclear counterstain and anti‐kinetochore antibodies, respectively, to score MN in binuclear or multinuclear cells and to differentiate compound‐induced MN by the presence of kinetochores. First, a reference data set was created by manual scoring using two clastogens and aneugens. After developing the automated scoring process, a set of 14 reference genotoxicants were studied. The automated image analysis yielded the expected results: 5/5 clastogens and 6/6 aneugens (sensitivity: 100%) as well as 3/3 non‐genotoxicants (specificity: 100%) were correctly identified. Further, a threshold was determined for identifying aneugens. Based on the data for our internally characterized reference compounds, unknown compounds that induce ≥53.8% k+ MN are classified as aneugens. The current data demonstrate excellent specificity and sensitivity and the methodology is superior to manual microscopic analysis in terms of speed and throughput as well as the absence of human bias. Environ. Mol. Mutagen. 60:227–242, 2019. © 2018 Wiley Periodicals, Inc.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Differentiation of Aneugens and Clastogens in the In Vitro Micronucleus Test by Kinetochore Scoring Using Automated Image Analysis

Wilde

Queisser

Holz³

et al. 2018

Environ and Mol Mutagen

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“…A MN is induced by direct DNA damage (clastogenicity) or by retardation of chromosome segregation (aneugenicity). Revealing the clastogenic or aneugenic mode of action (MoA) is important, especially in pharmaceutical development, which needs to define a threshold of genotoxicity [2]. In general, there is no genotoxicity threshold for clastogens, because direct interaction between a chemical and DNA cannot be ruled out even at very low exposure levels.…”

Section: Introductionmentioning

confidence: 99%

“…by ionizing radiation) but also by DNA single-strand breaks (SSB) after treatment with UV irradiation, topoisomerase inhibitors, and reactive oxygen species [6, 7]. The combination of MN test and γH2AX detection on a high-throughput platform will make it possible to discriminate aneugens from clastogens in the early screening stages because it was reported that aneugens do not form γH2AH foci [2] whereas clastogens do. Furthermore, imaging analysis makes it possible to morphologically discriminate γH2AX foci from pan-nucleic γH2AX staining, which is believed to be induced by non-genotoxic events, such as apoptosis [8, 9].…”

Section: Introductionmentioning

confidence: 99%

High-content imaging analyses of γH2AX-foci and micronuclei in TK6 cells elucidated genotoxicity of chemicals and their clastogenic/aneugenic mode of action

Takeiri¹,

Matsuzaki²,

Motoyama³

et al. 2019

Genes and Environ

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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.Electronic supplementary materialThe online version of this article (10.1186/s41021-019-0117-8) contains supplementary material, which is available to authorized users.

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“…Recently, γH2AX is also used as a marker of genotoxicity in pharmaceutical drug development [ 9 ]. Because the early stage of drug development requires high-throughput screening (HTS) assays that can rapidly evaluate a variety of chemical candidates, the simple methodology of the γH2AX assay makes it well-matched to this purpose.…”

Section: Introductionmentioning

confidence: 99%

Advantages of evaluating γH2AX induction in non-clinical drug development

Motoyama¹,

Takeiri²,

Tanaka³

et al. 2018

Genes and Environ

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γH2AX, the phosphorylated form of a histone variant H2AX at Ser 139, is already widely used as a biomarker to research the fundamental biology of DNA damage and repair and to assess the risk of environmental chemicals, pollutants, radiation, and so on. It is also beginning to be used in the early non-clinical stage of pharmaceutical drug development as an in vitro tool for screening and for mechanistic studies on genotoxicity. Here, we review the available information on γH2AX-based test systems that can be used to develop drugs and present our own experience of practically applying these systems during the non-clinical phase of drug development. Furthermore, the potential application of γH2AX as a tool for in vivo non-clinical safety studies is also discussed.

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Chromosomal aberrations clastogens vs aneugens

Cited by 31 publications

References 115 publications

Differentiation of Aneugens and Clastogens in the In Vitro Micronucleus Test by Kinetochore Scoring Using Automated Image Analysis

Differentiation of Aneugens and Clastogens in the In Vitro Micronucleus Test by Kinetochore Scoring Using Automated Image Analysis

High-content imaging analyses of γH2AX-foci and micronuclei in TK6 cells elucidated genotoxicity of chemicals and their clastogenic/aneugenic mode of action

Advantages of evaluating γH2AX induction in non-clinical drug development

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