High-specificity and high-sensitivity genotoxicity assessment in a human cell line: Validation of the GreenScreen HC GADD45a-GFP genotoxicity assay

Hastwell, Paul W.; Chai, Li Leng; Roberts, Kevin J.; Webster, Thomas W.; Harvey, James S.; Rees, Robert W.; Walmsley, Richard M.

doi:10.1016/j.mrgentox.2006.04.011

Cited by 160 publications

(123 citation statements)

References 39 publications

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“…The advantage of this is that cell processing occurs very efficiently, as all centrifugation steps can be eliminated. On the other hand, TK6 cells have the advantage of being of human origin, have a proficient p53 status and offer the possibility for combinational assays such as GreenScreen HC [30] or gene mutation analysis at the TK locus.…”

Section: Compatibility With Other Cell Linesmentioning

confidence: 99%

Interlaboratory evaluation of a flow cytometric, high content in vitro micronucleus assay

Bryce

Avlasevich

Bemis

et al. 2008

Mutation Research/Genetic Toxicology and Environmental Mutagene

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An international, multi-lab trial was conducted to evaluate a flow cytometry-based method for scoring micronuclei in mouse lymphoma L5178Y cells [Avlasevich et al., Environ. Molec. Mutagen. 47 (2006) [56][57][58][59][60][61][62][63][64][65][66]. A reference laboratory investigated the potential of six chemicals to induce micronuclei-the genotoxicants mitomycin C, etoposide, and vinblastine, and the non-genotoxicants sucrose, staurosporine, or dexamethasone. The latter two non-genotoxicants were selected as extreme challenges to the assay because of their potent apoptogenic activity. Three collaborating laboratories were supplied with prototype In Vitro MicroFlow ™ kits, and each was assigned one genotoxicant and one non-genotoxicant. Cells were treated continuously for 24 hrs over a range of concentrations up to 5 mg/ml, or overtly cytotoxic concentrations. Micronuclei were scored via standard microscopy and flow cytometry. In addition to enumerating micronucleus frequencies, a cytotoxicity measurement that is simultaneously acquired with the flow cytometric micronucleus scoring procedure was evaluated (Flow-NBR). With this method, latex particles served as counting beads, and facilitated relative survival measurements that exclude the presence of dead/dying cells. For comparison purposes, additional cytotoxicity endpoints were measured, including several that are based on cell number, and others that reflect compromised membrane integrity, including dye permeability and/or phospholipid distribution. Key findings for this set of compounds include the following: (1) significant discrepancies in top concentration selection were found when cytotoxicity measurements were based on different methods, with the Flow-NBR approach tending to be the most sensitive, (2) both microscopy-and flow cytometry-based scoring methods detected concentrationdependent micronucleus formation for the three genotoxic agents studied, with good agreement between the reference laboratory and the collaborating laboratories, and (3) whereas flow cytometric analyses showed no significant increases for the non-genotoxicants when top concentration selection was based on Flow-NBR, significantly elevated micronucleus frequencies were observed for concentrations that were chosen based on less-sensitive cytotoxicity assays. Collectively, these results indicate that rapid assessment of genotoxicity can be accomplished with a relatively simple Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access

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Section: Compatibility With Other Cell Linesmentioning

confidence: 99%

Interlaboratory evaluation of a flow cytometric, high content in vitro micronucleus assay

Bryce

Avlasevich

Bemis

et al. 2008

Mutation Research/Genetic Toxicology and Environmental Mutagene

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“…The choice of the pathways was mostly based on microarray experiments with genotoxic substances. The GreenScreen HC assay, which uses p53-competent TK6 lymphoblastoid cell line genetically modified to incorporate a fusion cassette containing the GADD45alfa promoter (and other regulatory elements) and the GFP gene as reporter (Hastwell et al, 2006), has been widely characterised and its good sensitivity and specificity confirmed in independent studies (Hastwell et al, 2009;Jagger et al, 2009;Birrel et al, 2010). HTS luciferase reporter assays based on four different stress pathways (RAD51C, Cystatin A, p53 and Nrf2) in the HepG2 cell line have also been developed and shown to be useful for pre-screening in early phases of drug development (Westerink et al, 2010).…”

Section: Genotoxicity Assays Based On Induction Of Dna Damage Responsmentioning

confidence: 99%

Scientific opinion on genotoxicity testing strategies applicable to food and feed safety assessment

2011

EFS2

285

136

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The Scientific Committee reviewed the current state-of-the-science on genotoxicity testing and provided a commentary and recommendations on genotoxicity testing strategies. A step-wise approach is recommended for the generation and evaluation of data on genotoxic potential, beginning with a basic battery of in vitro tests, comprising a bacterial reverse mutation assay and an in vitro micronucleus assay. Consideration should be given to whether specific features of the test substance might require substitution of one or more of the recommended in vitro tests by other in vitro or in vivo tests in the basic battery. In the event of negative in vitro results, it can be concluded that the substance has no genotoxic potential. In case of inconclusive, contradictory or equivocal results, it may be appropriate to conduct further testing in vitro. In case of positive in vitro results, review of the available relevant data on the test substance and, where necessary, an appropriate in vivo study to assess whether the genotoxic potential observed in vitro is expressed in vivo is recommended. Suitable in vivo tests are the mammalian erythrocyte micronucleus test, transgenic rodent assay, and Comet assay. The approach to in vivo testing should be step-wise. If the first in vivo test is positive, no further testing is necessary and the substance should be considered as an in vivo genotoxin. If the test is negative, it may be possible to conclude that the substance is not an in vivo genotoxin. However, in some cases, a second in vivo test may be necessary (e.g. if the first test is negative but more than one endpoint in the in vitro tests are positive, an in vivo test on a second endpoint may be necessary). The combination of assessing different endpoints in different tissues in the same animal in vivo should also be considered.

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“…GreenScreen GC, a new, high-throughput assay that links the regulation of the human GADD45a gene to the production of Green Fluorescent Protein (GFP) has become available. The assay relies on the DNA damage-induced up-regulation of the RAD54 gene in yeast measured using a promoter-GFP fusion reporter (44). The test is more specific and sensitive for genotoxicity than those currently recommended by the FDA such as the Ames and mouse lymphoma tests.…”

Section: Genotoxicitymentioning

confidence: 99%

De-Risking Drug Discovery Programmes Early with ADMET

Tsaioun¹,

Kates²

2011

Drug Discovery and Development - Present and Future

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High-specificity and high-sensitivity genotoxicity assessment in a human cell line: Validation of the GreenScreen HC GADD45a-GFP genotoxicity assay

Cited by 160 publications

References 39 publications

Interlaboratory evaluation of a flow cytometric, high content in vitro micronucleus assay

Interlaboratory evaluation of a flow cytometric, high content in vitro micronucleus assay

Scientific opinion on genotoxicity testing strategies applicable to food and feed safety assessment

De-Risking Drug Discovery Programmes Early with ADMET

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