Genetic modification and variations in solvent increase the sensitivity of the yeast RAD54-GFP genotoxicity assay.
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.
Faculty of Life Sciences, The Mill, University of Manchester, Sackville Street, Manchester M60 1QD, UK.
L
L
Walsh
P W
PW
Hastwell
P O
PO
Keenan
A W
AW
Knight
N
N
Billinton
R M
RM
Walmsley
D018528:ATP-Binding Cassette Transporters / Q000235:genetics; D002473:Cell Wall / Q000235:genetics; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics; D004265:DNA Helicases; D004260:DNA Repair / Q000235:genetics*; D045643:DNA Repair Enzymes; D000431:Ethanol / Q000737:chemistry; D017353:Gene Deletion; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics*; D006801:Humans; D009152:Mutagenicity Tests / Q000379:methods*; D009153:Mutagens / Q000633:toxicity; D009154:Mutation; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics*; D012680:Sensitivity and Specificity; D012997:Solvents / Q000737:chemistry
D018528:ATP-Binding Cassette Transporters; D009153:Mutagens; D029701:Saccharomyces cerevisiae Proteins; D012997:Solvents; D049452:Green Fluorescent Proteins; D000431:Ethanol; D003577:Cytochrome P-450 Enzyme System; C104464:CYP3A protein, human; D051544:Cytochrome P-450 CYP3A; C075468:RAD54 protein, S cerevisiae; D004265:DNA Helicases; D045643:DNA Repair Enzymes