The GreenScreen(R) genotoxicity assay: a screening validation programme

Abstract

A yeast (Saccharomyces cerevisiae) DNA repair reporter assay termed the GreenScreen assay (GSA) is described. This is a novel, cost-effective genotoxicity screen, developed to provide a pre-regulatory screening assay for use by the pharmaceutical industry and in other applications where significant numbers of compounds need to be tested. It provides a higher throughput and a lower compound consumption than existing eukaryotic genotoxicity assays and is sensitive to a broad spectrum of mutagens and, importantly, clastogens. We describe a simple, robust assay protocol and a validation study. The end-point of the test reflects the typically eukaryotic chromosomes and DNA metabolizing enzymes of yeast. The capacity for metabolic activation (MA) in yeast is limited compared with the mammalian liver or its extracts, but the assay does detect a subset of compounds that would require MA in existing genotoxicity tests. The GSA detects a different spectrum of compounds to bacterial genotoxicity assays and thus, together with an in silico structure-activity relationship (SAR) screen, and possibly a high throughput bacterial screen, would provide an effective preview of the regulatory battery of genotoxicity tests.

Construction and evaluation of two biosensors based on yeast transcriptional response to genotoxic chemicals

“…We also compared the HUG1-yEGFP and RNR3-yEGFP biosensors with an existing GreenScreen assay based on the budding yeast RAD54-GFP reporter (Cahill et al, 2004), which is summarized in Supplementary Table S3. The HUG1-yEGFP biosensor is about tenfold less sensitive to detect MV than the GreenScreen assay and is comparable for the detection of MMS.…”

Enhanced Cellular Uptake and Cytotoxicity Studies of Organometallic Bioconjugates of the NLS Peptide in Hep G2 Cells

“…Briefly, this test is based on yeast strains that carry reporter plasmids that express a green fluorescent protein (yEGFP) under the control of the promoters for the genes RNR2 and RAD54, which are induced in response to genotoxic DNA damage, and a control strain in which GFP is not expressed due to a frameshift mutation. [48,49] The tests were performed in black 96-well microplates for optimal fluorescence detection. All substances were tested in serial dilutions by starting from 2 mm stock solutions of the test compounds (in 4 % (v/v) aqueous DMSO); the final concentration ranged from 1.0 mm to 0.002 mm.…”

A pilot study for construction of a new cadmium-sensing yeast strain carrying a reporter plasmid with the <i>JLP1</i> promoter

“…The promoter sequences of RAD51, RAD54, HUG1, RNR2, and RNR3 have been used in reporter plasmids to detect agents that damage DNA, and assay strains have been shown to respond to various genotoxic agents such as gamma ray, methyl methanesulfonate, 4-nitroquinoline-1-oxide, methyl-Nnitro-N-nitrosoguanidine, cisplatin, and camptothecin (Walmsley et al, 1997;Billinton et al, 1998;Afanassiev et al, 2000;Boronat and Piña, 2006;Benton et al, 2007;Liu et al, 2008;Wei et al, 2013). The RAD54-GFP reporter assay strain has been validated as a genotoxicity test called the GreenScreen Assay (Cahill et al, 2004;Walsh et al, 2005;Van Gompel et al, 2005;Knight et al, 2007). The promoter sequences of the CUP1, SEO1, and UFO1 genes have been used to detect heavy metals.…”