Genetic modification and variations in solvent increase the sensitivity of the yeast RAD54-GFP genotoxicity assay

L. Walsh, P. W. Hastwell, P. O. Keenan, A. W. Knight, N. Billinton, R. M. Walmsley

    Research output: Contribution to journalArticlepeer-review

    Abstract

    The yeast (Saccharomyces cerevisiae) RAD54-GFP DNA repair reporter assay (GreenScreen® assay, GSA) can be used for early genotoxicity screening in drug discovery. During the initial validation of this preregulatory assay, a subset of known genotoxic compounds that did not give reproducibly clear positive GSA results was identified. Cell permeability, inherent drug resistance mechanisms, metabolic activation and compound solubility were identified as possible barriers to the detection of specific compounds. In this study three types of modification to the existing assay protocol were explored in order to address these possibilities: (i) modification of the reporter host strain by deletion of genes involved in cell wall integrity or with products functioning as efflux pumps (PDR5, ERG6, SNQ2, YOR1); (ii) expression in the host yeast of human phase I metabolic activation genes and (iii) variation in the test solvent system for compounds with poor aqueous solubility. The modifications described and the assay results presented show how the assay may be tailored to suit specific classes of test compound in a more analytical mode. Improvements in assay sensitivity were seen in the detection of some genotoxins using yeast cell wall mutants and those expressing human cytochrome P450 genes. © The Author 2005. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved.
    Original languageEnglish
    Pages (from-to)317-327
    Number of pages10
    JournalMutagenesis
    Volume20
    Issue number5
    DOIs
    Publication statusPublished - Sept 2005

    Fingerprint

    Dive into the research topics of 'Genetic modification and variations in solvent increase the sensitivity of the yeast RAD54-GFP genotoxicity assay'. Together they form a unique fingerprint.

    Cite this