Interlaboratory assessment of the GreenScreen HC GADD45a-GFP genotoxicity screening assay: An enabling study for independent validation as an alternative method

Billinton, Nicholas; Hastwell, Paul W.; Beerens, D.; Birrell, Louise; Ellis, Patricia; Maskell, Sharon; Webster, Thomas W.; Windebank, Sam; Woestenborghs, Filip; Lynch, Anthony M.; Scott, Andrew D.; Tweats, David; Gompel, Jacky Van; Rees, Robert W.; Walmsley, Richard M.

doi:10.1016/j.mrgentox.2008.02.011

Cited by 36 publications

(20 citation statements)

References 52 publications

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“…9 It has a high sensitivity (87%), detecting all classes of genotoxin: mutagens, promutagens, clastogens, and aneugens. 9,10 Specificity is a key characteristic for screening, and the assay's specificity (95%) 9,10 is higher than those historically reported for Ames 3 (sensitivity 60.3%, specificity 77.3%), the Micronucleus Test (MNT: 13,14 GFP reporters enable the development of simple, reagentfree assays and, partly as a consequence of this, are more amenable to moderate-throughput screening applications than the earlier generation of antibiotic resistance and other enzyme-linked reporter genes. There are, however, some applications in which assay users are prepared to accept additional handling steps if a higher signal-to-noise ratio can be obtained.…”

Section: Introductionmentioning

confidence: 92%

Development of a High-Throughput Gaussia Luciferase Reporter Assay for the Activation of the GADD45a Gene by Mutagens, Promutagens, Clastogens, and Aneugens

et al. 2012

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Exposure to genotoxic carcinogens leads to increased expression of the GADD45a gene in mammalian cells. This signature of genotoxic hazard has previously been exploited in the GreenScreen HC assay, in which GADD45a expression is linked to green fluorescent protein (GFP) expression in the human TK6 lymphoblastoid cell line. This article describes the development and validation of an alternative assay ("BlueScreen HC"), in which expression is linked to Gaussia luciferase (GLuc) expression, yielding a luminescent reporter, the preferred optical output in high-throughput screening. The coelentrazine substrate of GLuc is relatively unstable, and a new buffer is reported that improves its stability. A more sensitive method is demonstrated for the measurement of cell densities in the assay, using the fluorescent cyanine dye thiazole orange. A protocol amendment also allows the assessment of pro-genotoxicity using S9 liver extracts. Compounds from the European Centre for the Validation of Alternative Methods (ECVAM) recommended list for the assessment of new or improved genotoxicity assays were evaluated with and without S9 in the new assay. The new GLuc assay was as effective as the GFP assay in producing positive results for all classes of genotoxic carcinogen and negative results for all nongenotoxins tested.

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Section: Introductionmentioning

confidence: 92%

Development of a High-Throughput Gaussia Luciferase Reporter Assay for the Activation of the GADD45a Gene by Mutagens, Promutagens, Clastogens, and Aneugens

et al. 2012

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“…For example, BlueScreen TM test (Gentronix, Manchester, UK) is a screening tool currently being used in this capacity. This assay provides information on multiple genotoxic mechanisms and can be performed with and without metabolic activation (Billinton et al, 2008, Birrell et al, 2010, Hastwell et al, 2006, Hastwell et al, 2009, Knight et al, 2009, Hughes et al, 2012. Following this evaluation step the material moves into…”

Section: A Genotoxicitymentioning

confidence: 99%

Criteria for the Research Institute for Fragrance Materials, Inc. (RIFM) safety evaluation process for fragrance ingredients

Api

Belsito

Bruze

et al. 2015

Food and Chemical Toxicology

1,516

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“…In contrast to the regulatory in vitro mammalian assays, the results demonstrate high specificity without compromising sensitivity. This 96-well microplate assay is becoming widely employed and its transfer to other laboratories has been systematically evaluated [67]. The format of the GADD45a-GFP assay is very similar to the yeast assay.…”

Section: Eukaryotic (Human Cells)mentioning

confidence: 99%

Genetic Toxicity:In VitroApproaches for Medicinal Chemists

Walmsley

Elder

2010

ADMET for Medicinal Chemists

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Interlaboratory assessment of the GreenScreen HC GADD45a-GFP genotoxicity screening assay: An enabling study for independent validation as an alternative method

Cited by 36 publications

References 52 publications

Development of a High-Throughput Gaussia Luciferase Reporter Assay for the Activation of the GADD45a Gene by Mutagens, Promutagens, Clastogens, and Aneugens

Development of a High-Throughput Gaussia Luciferase Reporter Assay for the Activation of the GADD45a Gene by Mutagens, Promutagens, Clastogens, and Aneugens

Criteria for the Research Institute for Fragrance Materials, Inc. (RIFM) safety evaluation process for fragrance ingredients

Genetic Toxicity:In VitroApproaches for Medicinal Chemists

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