title: Development of a highthroughput yeast-based assay for detection of metabolically activated genotoxins.
abstract: The potential genotoxicity of drug candidates is a serious concern during drug development. Therefore, it is important to assess the potential genotoxicity and mutagenicity of a compound early in the discovery phase of drug development. AMES Salmonella assay is the most widely used assay for the assessment of mutagenicity and genotoxicity. However, the AMES assay is not readily adaptable to highthroughput screening and several strains of Salmonella must be employed to ensure that different types of DNA damage can be studied. Therefore, an additional robust highthroughput genotoxicity screen would be of significant value in the early detection and elimination of genotoxicity. The complexity of DNA damage requires numerous cellular pathways, thus using single model organism to predict genotoxicity in early stage is challenging. Another critical component of such screens is that they incorporate the capability of metabolic activation to ensure that no genotoxic metabolites are generated. We have developed a novel highthroughput reporter assay for DNA repair that detects genotoxicity, and which incorporates metabolic activation. The assay has a low compound requirement as compared to Ames, and relies upon two different reporter genes cotransfected into a yeast strain. The gene encoding Renilla luciferase is fused to the constitutive 3-phosphoglycerate kinase (PGK1) promoter and integrated into the yeast genome to provide a control for cell numbers. The firefly luciferase gene is fused to the RAD51 (bacterial RecA homolog) promoter and used to report an increase in DNA repair activity. A dual luciferase assay is performed by measuring the firefly and Renilla luciferase activities in the same sample. The result is expressed as the ratio of the two luciferase activities; changes from the base level (control) are interpreted as induction of the RAD51 promoter and evidence of DNA repair activity in eukaryote cells due to DNA damage. The yeast dual luciferase reporter has been characterized with and without S-9 activation using positive and negative control agents. This assay is efficient, requires little time and low amounts of compound. The assay is compatible with metabolic activation, adaptable to a highthroughput platform, and yields data that accurately and reproducibly detects DNA damage. Whereas the normal yeast cell wall, plasma membrane composition and the presence of active transporters can prevent the entry or persistence of some compounds internally in yeast cells, our assay did show concordance with regulatory mutagenicity assays, many of which require metabolic activation and are poorly detected by bacterial mutagenicity assays. Although there were false negative results, in our hands this assay performs as well as or better than other commercially available genetox assays. Furthermore, the RAD51 gene is strongly inducible by homologous intrachromosomal recombination; thus this assay may provide a means to detect clastogens. The RAD51 promoter fused dual luciferase assay represents a valuable addition to the armamentarium for the early detection of genotoxic compounds.
authors.affiliation: Drug Metabolism, Pharmacokinetics, and Toxicology, Lexicon Pharmaceuticals, Inc., The Woodlands, TX 77381, USA. mliu@lexpharma.com
authors.forename: Xuemei
authors.initials: X
authors.lastname: Liu
authors.forename: Jeffrey A
authors.initials: JA
authors.lastname: Kramer
authors.forename: Jonathan C
authors.initials: JC
authors.lastname: Swaffield
authors.forename: Yi
authors.initials: Y
authors.lastname: Hu
authors.forename: Guixuan
authors.initials: G
authors.lastname: Chai
authors.forename: Alan G E
authors.initials: AG
authors.lastname: Wilson
mesh_terms: D000818:Animals; D004260:DNA Repair / Q000187:drug effects* / Q000235:genetics; D016681:Genome, Fungal / Q000235:genetics; D049410:Luciferases, Renilla / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics; D009152:Mutagenicity Tests / Q000379:methods; D009153:Mutagens / Q000378:metabolism / Q000494:pharmacology*; D011401:Promoter Regions, Genetic / Q000235:genetics; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics; D048790:Renilla; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D012680:Sensitivity and Specificity
chemical_list: D009153:Mutagens; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D049410:Luciferases, Renilla