Reduction of misleading (“false”) positive results in mammalian cell genotoxicity assays. III: Sensitivity of human cell types to known genotoxic agents

Fowler, Paul; Smith, Robert; Smith, Katie; Young, Jamie; Jeffrey, Laura; Carmichael, Paul L.; Kirkland, David; Pfuhler, Stefan

doi:10.1016/j.mrgentox.2014.03.001

Cited by 50 publications

(37 citation statements)

References 13 publications

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“…In this study, four chemicals (two aneugens and two clastogens) known to induce MN, as well as one negative control chemical, were evaluated for cytotoxicity and genotoxicity. In samples not exposed to Cyt-B, increases in cytotoxicity were observed with increasing chemical dose as expected and all top doses induced a cytotoxicity of >55 6 5%, similar to results presented in published literature (7,(48)(49)(50). In samples exposed to Cyt-B however, cytotoxicity values at most doses from all chemicals tested tended to be lower than when Cyt-B was not used.…”

Section: Discussionsupporting

confidence: 88%

“…The current OECD guideline for performing the in vitro MN assay permits the use of several cell lines, both human and rodent as well as primary human or other mammalian peripheral blood lymphocytes (12). Several studies have been performed to test the in vitro MN assay using a number of different cell types including human peripheral blood lymphocytes (14,49), Chinese Hamster Ovary cells (13), Chinese Hamster Lung cells (17), and mouse lymphoma L5178Y cells (16,48,62), demonstrating positive results for many test agents. Therefore, the assay should be performed on the ISX using additional cell types and chemical agents to develop applicable sample processing methods and to demonstrate the adaptability of the method.…”

Section: Future Directionsmentioning

confidence: 99%

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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: 88%

Section: Future Directionsmentioning

confidence: 99%

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

Rodrigues¹

2018

Cytometry Pt A

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“…Overall, the data suggests that the use of human cell lines may be preferable over rodent cell lines to help reduce potential misleading positive results in in vitro assay systems with certain types of chemical (this being consistent with the observations of Fowler et al [2,31,41] who concluded that the use of a human cell type with functional p53 could reduce the detection of misleading positive results). For these investigations clear induction of micronuclei was less prevalent when using human cell lines versus mouse for this set of chemicals.…”

Section: Discussionsupporting

confidence: 85%

Relationships between p53 status, apoptosis and induction of micronuclei in different human and mouse cell lines in vitro: Implications for improving existing assays

Whitwell

Smith

Jenner

et al. 2015

Mutation Research/Genetic Toxicology and Environmental Mutagene

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“…Consequently, human cell lines with altered responsiveness to DNA damaging mechanisms may be expected to generate results not dissimilar to those produced in rodent cell lines. At this time there are not enough data available to reliably determine if the use of p53-competent cell lines of human origin (as opposed to p53-competent rodent derived lines) or other human cells confer greater accuracy (Walmsley & Billinton 2011;Fowler et al 2014). …”

Section: Human Versus Non-human Test Resultsmentioning

confidence: 99%

Genotoxicity Expert Panel review: weight of evidence evaluation of the genotoxicity of glyphosate, glyphosate-based formulations, and aminomethylphosphonic acid

Brusick¹,

Aardema²,

Kier

et al. 2016

Critical Reviews in Toxicology

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published a monograph concluding there was strong evidence for genotoxicity of glyphosate and glyphosate formulations and moderate evidence for genotoxicity of the metabolite aminomethylphosphonic acid (AMPA). These conclusions contradicted earlier extensive reviews supporting the lack of genotoxicity of glyphosate and glyphosate formulations. The IARC Monograph concluded there was strong evidence of induction of oxidative stress by glyphosate, glyphosate formulations, and AMPA. The Expert Panel reviewed the genotoxicity and oxidative stress data considered in the IARC Monograph, together with other available data not considered by IARC. The Expert Panel defined and used a weight of evidence (WoE) approach that included ranking of studies and endpoints by the strength of their linkage to events associated with carcinogenic mechanisms. Importantly, the Expert Panel concluded that there was sufficient information available from a very large number of regulatory genotoxicity studies that should have been considered by IARC. The WoE approach, the inclusion of all relevant regulatory studies, and some differences in interpretation of individual studies led to significantly different conclusions by the Expert Panel compared with the IARC Monograph. The Expert Panel concluded that glyphosate, glyphosate formulations, and AMPA do not pose a genotoxic hazard and the data do not support the IARC Monograph genotoxicity evaluation. With respect to carcinogenicity classification and mechanism, the Expert Panel concluded that evidence relating to an oxidative stress mechanism of carcinogenicity was largely unconvincing and that the data profiles were not consistent with the characteristics of genotoxic carcinogens. ARTICLE HISTORY

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Reduction of misleading (“false”) positive results in mammalian cell genotoxicity assays. III: Sensitivity of human cell types to known genotoxic agents

Cited by 50 publications

References 13 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

Relationships between p53 status, apoptosis and induction of micronuclei in different human and mouse cell lines in vitro: Implications for improving existing assays

Genotoxicity Expert Panel review: weight of evidence evaluation of the genotoxicity of glyphosate, glyphosate-based formulations, and aminomethylphosphonic acid

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Reduction of misleading (“false”) positive results in mammalian cell genotoxicity assays. III: Sensitivity of human cell types to known genotoxic agents

Cited by 50 publications

References 13 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

Relationships between p53 status, apoptosis and induction of micronuclei in different human and mouse cell lines in vitro: Implications for improving existing assays

Genotoxicity Expert Panel review: weight of evidence evaluation of the genotoxicity of glyphosate, glyphosate-based formulations, and aminomethylphosphonic acid

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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