GADD45a-GFP GreenScreen HC assay results for the ECVAM recommended lists of genotoxic and non-genotoxic chemicals for assessment of new genotoxicity tests

Birrell, Louise; Cahill, Paul A.; Hughes, Chris; Tate, Matthew; Walmsley, Richard M.

doi:10.1016/j.mrgentox.2009.12.008

Cited by 52 publications

(37 citation statements)

References 45 publications

(30 reference statements)

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“…Such scenarios might lead to the generation of misleading negative data, although there have been no published reports of inhibitors associated with the use of GLuc. Comparison of the GLuc assay results in this study with those published for the GFP assay 12 suggests that chemical interference with GLuc may not be a common phenomenon, as most compounds produced the same results in the two assays. For this reason, only those compounds where results differ between the two assays are discussed further.…”

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confidence: 58%

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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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confidence: 58%

“…p-Nitrophenol was positive only in the GADD45a-GFP assay at the highest tolerated dose (10 mM) in the S9 protocol. 12 Eugenol was positive only in the GADD45a-GLuc assay and at the highest tolerated dose (-S9, 1.25 mM; +S9, 2.5 mM). Had these compounds been tested to the new recommended top concentration (1 mM), 1 all would have produced negative results.…”

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confidence: 93%

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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“…Hastwell et al 27 developed an assay that exploits a reporter system in which the expression of EGFP is controlled by regulatory elements of the GADD45a gene hosted in the p53-competent human lymphoblastoid TK6 cell line. A thorough validation of this assay showed its high sensitivity and specificity 28. The assay is commercially available as GreenScreen HC assay provided by Gentronics Ltd (UK).…”

Section: Discussionmentioning

confidence: 99%

Development of human cell biosensor system for genotoxicity detection based on DNA damage-induced gene expression

Žager¹,

Čemažar²,

Hreljac³

et al. 2010

Radiology and Oncology

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BackgroundHuman exposure to genotoxic agents in the environment and everyday life represents a serious health threat. Fast and reliable assessment of genotoxicity of chemicals is of main importance in the fields of new chemicals and drug development as well as in environmental monitoring. The tumor suppressor gene p21, the major downstream target gene of activated p53 which is responsible for cell cycle arrest following DNA damage, has been shown to be specifically up-regulated by genotoxic carcinogens. The aim of our study was to develop a human cell-based biosensor system for simple and fast detection of genotoxic agents.MethodsMetabolically active HepG2 human hepatoma cells were transfected with plasmid encoding Enhanced Green Fluorescent Protein (EGFP) under the control of the p21 promoter (p21HepG2GFP). DNA damage was induced by genotoxic agents with known mechanisms of action. The increase in fluorescence intensity, due to p21 mediated EGFP expression, was measured with a fluorescence microplate reader. The viability of treated cells was determined by the colorimetric MTS assay.ResultsThe directly acting alkylating agent methylmethane sulphonate (MMS) showed significant increase in EGFP production after 48 h at 20 μg/mL. The indirectly acting carcinogen benzo(a)pyren (BaP) and the cross-linking agent cisplatin (CisPt) induced a dose- dependent increase in EGFP fluorescence, which was already significant at concentrations 0.13 μg/mL and 0.41 μg/mL, respectively. Vinblastine (VLB), a spindle poison that does not induce direct DNA damage, induced only a small increase in EGFP fluorescence intensity after 24 h at the lowest concentration (0.1 μg/mL), while exposure to higher concentrations was associated with significantly reduced cell viability.ConclusionsThe results of our study demonstrated that this novel assay based on the stably transformed cell line p21HepG2GFP can be used as a fast and simple biosensor system for detection of genetic damage caused by chemical agents.

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“…This fluorescent reporter assay uses the GADD45α (growth arrest and DNA damage inducible alpha) gene promoter fused to a GFP marker for the detection of genotoxic agents in p53-competent human-derived TK6 cells (138). When testing the the European Centre for the Validation of Alternative Methods (ECVAM) recommended lists of genotoxic and non-genotoxic chemicals for assessment of new genotoxicity tests, the GreenScreen HC assays showed good sensitivity and specificity (139). More recently, the BlueScreen HC, a luciferase-based version of the GADD45α reporter assay, was described (140), and this assay has also been developed into a 384-well version (141).…”

Section: Modifications Of the Automated Fadu Assaymentioning

confidence: 99%

Emerging metrology for high-throughput nanomaterial genotoxicology

Nelson

Wright

Ibuki

et al. 2016

MUTAGE

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The rapid development of the engineered nanomaterial (ENM) manufacturing industry has accelerated the incorporation of ENMs into a wide variety of consumer products across the globe. Unintentionally or not, some of these ENMs may be introduced into the environment or come into contact with humans or other organisms resulting in unexpected biological effects. It is thus prudent to have rapid and robust analytical metrology in place that can be used to critically assess and/or predict the cytotoxicity, as well as the potential genotoxicity of these ENMs. Many of the traditional genotoxicity test methods [e.g. unscheduled DNA synthesis assay, bacterial reverse mutation (Ames) test, etc.,] for determining the DNA damaging potential of chemical and biological compounds are not suitable for the evaluation of ENMs, due to a variety of methodological issues ranging from potential assay interferences to problems centered on low sample throughput. Recently, a number of sensitive, high-throughput genotoxicity assays/platforms (CometChip assay, flow cytometry/micronucleus assay, flow cytometry/γ-H2AX assay, automated ‘Fluorimetric Detection of Alkaline DNA Unwinding’ (FADU) assay, ToxTracker reporter assay) have been developed, based on substantial modifications and enhancements of traditional genotoxicity assays. These new assays have been used for the rapid measurement of DNA damage (strand breaks), chromosomal damage (micronuclei) and for detecting upregulated DNA damage signalling pathways resulting from ENM exposures. In this critical review, we describe and discuss the fundamental measurement principles and measurement endpoints of these new assays, as well as the modes of operation, analytical metrics and potential interferences, as applicable to ENM exposures. An unbiased discussion of the major technical advantages and limitations of each assay for evaluating and predicting the genotoxic potential of ENMs is also provided.

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GADD45a-GFP GreenScreen HC assay results for the ECVAM recommended lists of genotoxic and non-genotoxic chemicals for assessment of new genotoxicity tests

Cited by 52 publications

References 45 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

Development of human cell biosensor system for genotoxicity detection based on DNA damage-induced gene expression

Emerging metrology for high-throughput nanomaterial genotoxicology

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