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Mutagenesis Advance Access published online on June 28, 2005
Mutagenesis, 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
Received March 3, 2005
Revised May 25, 2005
Accepted June 6, 2005

Original article

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

L. Walsh 1, P. W. Hastwell 2, P. O. Keenan 3, A. W. Knight 3, N. Billinton 3, and R. M. Walmsley 4*

1 Faculty of Life Sciences, The Mill, University of Manchester, Sackville Street, Manchester M60 1QD, UK; Present address: Department of Pharmacy and Pharmaceutical Sciences, Coupland 3 Building, University of Manchester, Oxford Road, Manchester M13 9PL, UK
2 Faculty of Life Sciences, The Mill, University of Manchester, Sackville Street, Manchester M60 1QD, UK
3 Gentronix Limited, The Fairbairn Building, 72 Sackville Street, Manchester M60 1QD, UK
4 Faculty of Life Sciences, The Mill, University of Manchester, Sackville Street, Manchester M60 1QD, UK; Gentronix Limited, The Fairbairn Building, 72 Sackville Street, Manchester M60 1QD, UK

* To whom correspondence should be addressed.
R. M. Walmsley, E-mail: richard.walmsley{at}manchester.ac.uk


  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.


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