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Mutagenesis Advance Access originally published online on June 28, 2005
Mutagenesis 2005 20(5):317-327; doi:10.1093/mutage/gei044
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© The Author 2005. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please email: journals.permissions@oupjournals.org

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

L. Walsh1,3, P.W. Hastwell1, P.O. Keenan2, A.W. Knight2, N. Billinton2 and R.M. Walmsley1,2,*

1Faculty of Life Sciences, The Mill, University of Manchester, Sackville Street, Manchester M60 1QD, UK and 2Gentronix Limited, The Fairbairn Building, 72 Sackville Street, Manchester M60 1QD, UK

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.

3 Present address: Department of Pharmacy and Pharmaceutical Sciences, Coupland 3 Building, University of Manchester, Oxford Road, Manchester M13 9PL, UK

* To whom correspondence should be addressed. Tel: +44 161 306 4174; Fax: +44 161 236 0409; Email: richard.walmsley{at}manchester.ac.uk


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