Comparison of four different treatment conditions of extended exposure in the in vitro micronucleus assay using TK6 lymphoblastoid cells

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

doi:10.1016/j.yrtph.2010.08.016

Cited by 16 publications

(14 citation statements)

References 27 publications

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“…Similar trends have been observed by Massey and Hinchliffe who reported several negative cytotoxicity values when using VS in V79 cells and by Elhajouji who demonstrated cytotoxicity values below 15% up to doses of 1.0 lg/mL of MMC in TK6 cells (7,52). Additionally, Hashimoto et al (53) observed opposing cytotoxicity trends in the presence and absence of Cyt-B using a number of clastogens and aneugens in TK6 cells, albeit with different exposure/recovery schedules. Exposure of cells to Mannitol showed an increase in cytotoxicity of <30% both in the presence and absence of Cyt-B.…”

Section: Discussionsupporting

confidence: 79%

Automation of the in vitro micronucleus assay using the Imagestream^® imaging flow cytometer

Rodrigues¹

2018

Cytometry Pt A

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The in vitro micronucleus (MN) assay is a well‐established test for evaluating genotoxicity and cytotoxicity. The use of manual microscopy to perform the assay can be laborious and often suffers from user subjectivity and interscorer variability. Automated methods including slide‐scanning microscopy and conventional flow cytometry have been developed to eliminate scorer bias and improve throughput. However, these methods possess several limitations such as lack of cytoplasmic visualization using slide‐scanning microscopy and the inability to visually confirm the legitimacy of MN or storage of image data for re‐evaluation using flow cytometry. The ImageStreamX® MK II (ISX) imaging flow cytometer has been demonstrated to overcome all of these limitations. The ISX combines the speed, statistical robustness, and rare event capture capability of conventional flow cytometry with high resolution fluorescent imagery of microscopy and possesses the ability to store all collected image data. This paper details the methodology developed to perform the in vitro MN assay in human lymphoblastoid TK6 cells on the ISX. High resolution images of micronucleated mono‐ and bi‐nucleated cells as well as polynucleated cells can be acquired at a high rate of capture. All images can then be automatically identified, categorized and enumerated in the data analysis software that accompanies the ImageStream, allowing for the scoring of both genotoxicity and cytotoxicity. The results demonstrate that statistically significant increases in MN frequency when compared with solvent controls can be detected at varying levels of cytotoxicity following exposure to well‐known aneugens and clastogens. This work demonstrates a fully automated method for performing the in vitro micronucleus assay on the ISX imaging flow cytometry platform. © 2018 The Author. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of ISAC.

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Section: Discussionsupporting

confidence: 79%

Automation of the in vitro micronucleus assay using the Imagestream^® imaging flow cytometer

Rodrigues¹

2018

Cytometry Pt A

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“…Others have shown that extending the recovery time for TK6 cells improves sensitivity in the micronucleus assay for some compounds. Specifically, when cells were continuously treated with MMC for 24 hr the resulting micronucleus frequency was approximately 5%, which increased to 20% when allowed a 24 hr recovery period [Hashimoto et al, ]. In contrast, cells treated with another alkylating agent, methylmethanesulfonate (MMS), did not show such a striking increase in micronucleus frequency with added recovery time (approximately 5% vs. 7%).…”

Section: Discussionmentioning

confidence: 99%

“…In contrast, cells treated with another alkylating agent, methylmethanesulfonate (MMS), did not show such a striking increase in micronucleus frequency with added recovery time (approximately 5% vs. 7%). In the case of non‐direct acting genotoxins, aphidicolin (polymerase inhibitor), and benomyl (aneugen) did not exhibit an increase in micronucleus induction with the added recovery time [Hashimoto et al, ]. Other compounds with varying mechanisms of genotoxicity were tested in the same study: bleomycin, cytosine arabinoside, hydroxyurea, and colcemid all caused an increase in micronucleus frequency when the recovery time was extended [Hashimoto et al, ].…”

Section: Discussionmentioning

confidence: 99%

“…In the case of non‐direct acting genotoxins, aphidicolin (polymerase inhibitor), and benomyl (aneugen) did not exhibit an increase in micronucleus induction with the added recovery time [Hashimoto et al, ]. Other compounds with varying mechanisms of genotoxicity were tested in the same study: bleomycin, cytosine arabinoside, hydroxyurea, and colcemid all caused an increase in micronucleus frequency when the recovery time was extended [Hashimoto et al, ]. Without accompanying cell cycle data it is not possible to determine whether cell cycle perturbations are responsible for the differences in micronucleus frequencies observed at early versus later time points.…”

Section: Discussionmentioning

confidence: 99%

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Impact of cell cycle delay on micronucleus frequency in TK6 cells

Sobol

Spellman

Thiffeault

et al. 2013

Environ and Mol Mutagen

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Previous studies with TK6 cells have shown that extending the recovery period after pulse treatment allows for greater micronucleus expression for some compounds. This study explores the role of cell cycle delay in micronucleus expression after pulse treatment with three model genotoxins [mitomycin C, etoposide (ETOP), vinblastine]. Cells were treated for 4 hr and allowed to recover for 36 hr with samples removed at various time points during the recovery period and analyzed for cell cycle distribution, apoptosis and micronucleus frequency. Our results show that mitomycin C causes cell cycle delay for 20 hr after pulse treatment and cell cycle perturbation is no longer evident after 36 hr of recovery. The micronucleus frequency of cells sampled at 36 hr is doubled when compared with cells sampled at 20 hr after mitomycin C removal. When cells were treated with indirect acting genotoxins (ETOP, vinblastine), cell cycle perturbation was not observed at the 20 hr time point. Micronucleus frequency after treatment with either ETOP or vinblastine did not differ between the 20 hr and the 36 hr time point. All three compounds induced similar levels of apoptosis ranging from 4.5 to 5.6% with maximum induction occurring at the 36-hr time point. We conclude that TK6 cells exhibit extended cell cycle arrest after exposure to MMC and can go on to express micronuclei, after overcoming cell cycle arrest.

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“…A minimum of 2,000 mononucleated cells from each dose level were scored for the presence of micronuclei using established criteria. 29 For the short-term exposure (4 hours), TK6 cells (2.5 Â 10 5 cells/mL) were treated with BH-BD in the absence or presence of S9. Following 4 hours exposure, cultures were washed, the treatment medium was replaced by CCM and cells were incubated for an additional 23 hours.…”

Section: Treatment Sampling and Analysismentioning

confidence: 99%

Genetic Toxicity Studies of the Ketogenic Ester Bis Hexanoyl (R)-1,3-Butanediol

Stubbs

Nikiforov²,

Rihner³

et al. 2021

Int J Toxicol

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A series of studies was conducted to assess the genetic toxicity of a novel ketone ester, bis hexanoyl (R)-1,3-butanediol (herein referred to as BH-BD), according to Organization for Economic Co-operation and Development testing guidelines under the standards of Good Laboratory Practices. In bacterial reverse mutation tests, there was no evidence of mutagenic activity in any of the Salmonella typhimurium strains tested or in Escherichia coli strain WP2 uvrA, at dose levels up to 5,000 μg/plate in the presence or absence of Aroclor 1254-induced rat liver (S9 mix) for metabolic activation. In the in vitro micronucleus test using human TK6 cells, BH-BD did not show a statistically significant increase in the number of cells containing micronuclei when compared with concurrent control cultures at all time points and at any of the concentrations analyzed (up to 100 μg/mL, final concentration in culture medium), with and without S9 mix activation. In the in vivo micronucleus test using Sprague Dawley rats, BH-BD did not show a statistically significant increase in the incidence of micronucleated polychromatic erythrocytes relative to the vehicle control group. Therefore, BH-BD was concluded to be negative in all 3 tests. These results support the safety assessment of BH-BD for potential use in food.

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Comparison of four different treatment conditions of extended exposure in the in vitro micronucleus assay using TK6 lymphoblastoid cells

Cited by 16 publications

References 27 publications

Automation of the in vitro micronucleus assay using the Imagestream^® imaging flow cytometer

Automation of the in vitro micronucleus assay using the Imagestream^® imaging flow cytometer

Impact of cell cycle delay on micronucleus frequency in TK6 cells

Genetic Toxicity Studies of the Ketogenic Ester Bis Hexanoyl (R)-1,3-Butanediol

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Comparison of four different treatment conditions of extended exposure in the in vitro micronucleus assay using TK6 lymphoblastoid cells

Cited by 16 publications

References 27 publications

Automation of the in vitro micronucleus assay using the Imagestream® imaging flow cytometer

Automation of the in vitro micronucleus assay using the Imagestream® imaging flow cytometer

Impact of cell cycle delay on micronucleus frequency in TK6 cells

Genetic Toxicity Studies of the Ketogenic Ester Bis Hexanoyl (R)-1,3-Butanediol

Contact Info

Product

Resources

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Automation of the in vitro micronucleus assay using the Imagestream^® imaging flow cytometer

Automation of the in vitro micronucleus assay using the Imagestream^® imaging flow cytometer