Molecular toxicology is playing an increasing role in the early development of pharmaceuticals, cosmetics, chemicals, and food additives. There is a continuing need for high-throughput genetic toxicology assays more predictive of carcinogenicity than currently available assays and regulatory agencies including FDA and EPA are increasingly accepting data from novel assays which give biological and mechanistic insights applicable to risk assessment. Assays for deletions (DEL) in yeast, human cells and in vivo in mice detect Salmonella (Ames assay) positive as well as Salmonella-negative carcinogens. The long-term objective of this proposal is to develop a new, rapid, high throughput DEL assay in mammalian cells as a commercially viable screen for potential carcinogens. For Phase lit is proposed to construct an Hprt-GFP fusion gene whose product emits fluorescence, and to start construction of an HprtGFP deletion-recombination construct. We will investigate the feasibility of fluorescence microscopy and flow cytometry for quantification of low frequency recombination events and begin to design and evaluate new integrated hardware and software platforms for assay automation. In Phase II we will finish construction of the Hprt duplication, generate DEL cell lines, implement and refine automated methods and validate the assay with 50 chemicals of known carcinogenic activity.
There is great interest within the pharmaceutical, cosmetic and chemical industries for a sensitive, high-throughput assay predictive of carcinogenesis, which could be used at an early stage in compound development, thereby reducing development time and cost.
