High-specificity and high-sensitivity genotoxicity assessment in a human cell line: validation of the GreenScreen HC GADD45a-GFP genotoxicity assay.
The battery of genetic toxicity tests required by most regulatory authorities includes both bacterial and mammalian cell assays and identifies practically all genotoxic carcinogens. However, the relatively high specificity of the Salmonella mutagenicity assay (Ames test) is offset by the low specificity of the established mammalian cell assays, which leads to difficulties in the interpretation of the biological relevance of results. This paper describes a new high-throughput assay that links the regulation of the human GADD45a gene to the production of Green Fluorescent Protein (GFP). A study of 75 well-characterised genotoxic and non-genotoxic compounds with diverse mechanisms of DNA-damage induction (including aneugens) reveals that the assay responds positively to all classes of genotoxic damage with both high specificity and high sensitivity. The current micro-well assay format does not include metabolic activation, but a separate low-throughput protocol demonstrates a successful proof-of-principle for an S9 metabolic activation assay with the model pro-mutagen cyclophosphamide. The test should be of value both as a tool in the selection of candidate compounds for further development, where additional data may be required because of conflicting information from the in vitro test battery, or in product development areas where the use of animals is to be discontinued. As a microplate assay however, it has the qualities of high throughput and low compound use that will facilitate its application in early screening for genotoxic liability.
Hastwell
Paul W
PW
Faculty of Life Sciences, University of Manchester, Manchester M60 1QD, UK.
Chai
Li-Leng
LL
Roberts
Kevin J
KJ
Webster
Thomas W
TW
Harvey
James S
JS
Rees
Robert W
RW
Walmsley
Richard M
RM
D001681:Biological Assay / Q000592:standards; D018797:Cell Cycle Proteins / Q000235:genetics* / Q000633:toxicity*; D002460:Cell Line; D004353:Drug Evaluation, Preclinical; D005786:Gene Expression Regulation / Q000187:drug effects; D005809:Genes, Regulator / Q000187:drug effects; D017930:Genes, Reporter / Q000187:drug effects; D049452:Green Fluorescent Proteins / Q000096:biosynthesis*; D006801:Humans; D066298:In Vitro Techniques; D009152:Mutagenicity Tests / Q000592:standards*; D009153:Mutagens / Q000633:toxicity*; D009687:Nuclear Proteins / Q000235:genetics* / Q000633:toxicity*; D011237:Predictive Value of Tests; D015203:Reproducibility of Results; D012107:Research Design; D012680:Sensitivity and Specificity; D000097642:GADD45 Proteins
D018797:Cell Cycle Proteins; D049452:Green Fluorescent Proteins; D009153:Mutagens; D009687:Nuclear Proteins; C439070:GADD45A protein, human; D000097642:GADD45 Proteins