Validation of Gene Expression Based Toxicity Screening

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DESCRIPTION (provided by applicant): The overall goal of this proposal is to provide a pathway for translating microarray-based research in cancer and toxicology into cost efficient, high throughput, validated assays for the assessment of a range of different toxicological and oncological mechanistic endpoints. Central to this proposal is the use of a highly multiplexed, quantitative, rtPCR-based gene expression analysis method that we have developed in our lab. The expression profiling (XP) methodology incorporates the use of gene-specific and universal primers to lock gene ratios during PCR amplification and is generally used to amplify multiplexes containing 20-30 genes per reaction. Advantages of the method include (a) low cost per assay via the use of standard, off-the-shelf reagents and equipment, (b) high throughput of >1000 samples per day, more with automation, and (c) high sensitivity, detecting single copy per cell transcripts using as little as 5 ng of total RNA per reaction. The use of off-the-shelf reagents and equipment also means that the assay can be readily performed in a range of academic, corporate and clinical settings. Our group proposes to identify and validate a set of genes that can be used for detailed toxicological evaluation of new chemotherapy and chemopreventive agents. In the Phase I of this proposed work, we intend to focus on the development and validation of gene sets for two specific toxicological endpoints, genotoxic and non-genotoxic carcinogenicity. The process will involve the performance of dose response studies with primary rat hepatocytes and a set of approximately 80 compounds with known genotoxic and non-genotoxic activities. The XP method will be used to monitor the expression levels of approximately 100 genes that are known to be involved in response to toxins. Analysis of these gene data will be used to identify a smaller set of genes that are predictive of genotoxic and non-genotoxic activities.