An in vitro micronucleus assay with size-classified micronucleus counting to discriminate aneugens from clastogens

Hashimoto, Kiyohiro; Nakajima, Y.; Matsumura, Shigeo; Chatani, Fumio

doi:10.1016/j.tiv.2009.09.006

Cited by 70 publications

(59 citation statements)

References 23 publications

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“…Differing from the results obtained for the other genotoxic agents employed in the present study, GF resulted less cytotoxic and genotoxic for neurons than for glial cells, with more accused decrease in viability, and dose-dependent increases in MN formation just observed in A172 cells. As aneugenicity caused by GF does not correlate with gene mutation or clastogenicity induction that results in chromosome aberrations via damage to DNA strands (Hashimoto et al, 2010), the slight positive effects observed in comet and gH2AX assays are likely due to cytotoxicity. To the best of our knowledge, there are no reports on GF neurogenotoxicity in the literature.…”

Section: Discussionmentioning

confidence: 94%

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Comparative study of human neuronal and glial cell sensitivity for in vitro neurogenotoxicity testing

Laffón

Fernández-Bertólez

Costa

et al. 2017

Food and Chemical Toxicology

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a b s t r a c tCell cultures from neuronal and glial origin have proven to be powerful tools for elucidating cellular and molecular mechanisms of nervous system development and physiology, and as neurotoxicity models to evaluate in vitro the possible effects of chemicals. But cellular heterogeneity of nervous system is considerable and these cells have been shown to respond diversely to neurotoxic insults, leading to disparate results from different studies. To shed more light on suitability of cellular models of nervous origin for neurotoxicity screening, the objective of this study was to compare the sensitivity to genetic damage induction of two nervous cell lines. To this aim, neurons (SH-SY5Y) and glial (A172) cells were treated with differently-acting genotoxic agents (bleomycin, actinomycin-D, methyl methanesulfonate, mitomycin C, and griseofulvin). After discarding cytotoxicity, genotoxicity was evaluated by a battery of assays encompassing detection of different genetic lesions. Results obtained showed that glial cells are generally more resistant to genotoxic damage induced by clastogenic agents, but more sensitive to aneugenic effects. These results highlight the need of proper design of in vitro neurotoxicology studies, especially for neurogenotoxicity screening, emphasizing the importance of employing more than one nervous cell type for testing the potential toxicity of a particular exposure.

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Section: Discussionmentioning

confidence: 94%

“…employed as positive control in genotoxicity assessment (Hashimoto et al, 2010;Porcedda et al, 2006;Sanchez-Flores et al, 2015). In a previous study with primary rat neurons, it was reported that cells remained alive and intact after 48 h of 0.1 mg/ml Act-D treatment (Martin et al, 1988).…”

Section: Discussionmentioning

confidence: 99%

Comparative study of human neuronal and glial cell sensitivity for in vitro neurogenotoxicity testing

Laffón

Fernández-Bertólez

Costa

et al. 2017

Food and Chemical Toxicology

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“…The calculation of MN size was also used as an additional parameter to indicate whether the activity of the tested substances was clastogenic or aneugenic [19,26]. Small size MN is more likely to contain acentric chromosome fragments indicating a clastogenic effect, while large size MN may possibly contain whole chromosomes thus indicating an aneugenic effect [27][28][29].…”

Section: Cbmn Assay In Human Lymphocytes In Vitromentioning

confidence: 99%

“…The size ratio of MN in the in vitro CBMN assay is an alerting index as effective as the fluorescence in situ hybridization (FISH) analysis for the discrimination of clastogenic and aneugenic effects [19,26].…”

Section: Cbmn Assay In Human Lymphocytes In Vitromentioning

confidence: 99%

Genotoxicity study of photolytically treated 2-chloropyridine aqueous solutions

Vlastos

Skoutelis

Theodoridis

et al. 2010

Journal of Hazardous Materials

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“…However, these assays are time consuming and typically not suitable to differentiate aneuploidy from clastogenicity for dozens of compounds in a particular chemical series [Eastmond and Tucker, 1989;Krishna et al, 1992]. Work has been done on multiple fronts to use MN sizing to distinguish whole from partial chromosomes within a standard MNvit assay [Hashimoto et al, 2010], and to incorporate FISH and centromeric or kinetochore probes to flow cytometry methods [Elhajouji et al, 1995;Miller and Nusse, 1993].…”

Section: Introductionmentioning

confidence: 99%

In vitro micronucleus screening of pharmaceutical candidates by flow cytometry in Chinese hamster V79 cells

Nicolette

Diehl

Sonders

et al. 2010

Environ and Mol Mutagen

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We previously reported a high concordance of in vitro micronucleus (MNvit) results obtained by flow cytometry to the known cytogenetic activity often commercially available compounds mentioned as validation compounds in an early draft of the OECD MNvit TG487 [Bryce et al., 2010; Organization for Economic Co-operation and Development(OECD), 2007]. The current study investigated this method in Chinese hamster V79 cells with pharmaceutical compounds of unknown genotoxic potential. Twenty-five compounds from several therapeutic areas such as oncology, neuroscience and immunological research were tested in the flow cytometry assay, and for comparison using the cytokinesis-block microscopy assay. Five of these 25 compounds were considered positive for micronucleus induction by the microscopy assessment. In all cases, the results from the flow cytometry assess ment matched the results of the microscopy assay. Thus, flow cytometry is a viable method for assessing the aneugenic/clastogenic potential of pharmaceutical drug candidates. The flow method offered several advantages over traditional microscopy. For instance, the ratio of micronuclei (MN) to 10,000 nuclei was evaluated in less than 2 min vs.15 min to manually assess 600 binucleate cells. Evaluation by flow cytometry can be automated,freeing resources and eliminating scorer fatigue.The assay may also provide for mechanistic understanding of MN formation based on size and the ratio of nuclei with sub-2N DNA content, allowing for discrimination between aneugenic and clastogenic compounds.

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An in vitro micronucleus assay with size-classified micronucleus counting to discriminate aneugens from clastogens

Cited by 70 publications

References 23 publications

Comparative study of human neuronal and glial cell sensitivity for in vitro neurogenotoxicity testing

Comparative study of human neuronal and glial cell sensitivity for in vitro neurogenotoxicity testing

Genotoxicity study of photolytically treated 2-chloropyridine aqueous solutions

In vitro micronucleus screening of pharmaceutical candidates by flow cytometry in Chinese hamster V79 cells

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