This Phase I proposal will develop and validate assays to measure the metabolism of compounds and their affect on liver cells using a multiplexed gene expression assay platform, the quantitative Nuclease Protection Assay (qNAP?) that can measure the expression of up to 100 mRNA and miRNA and is a simple, robust and highly precise assay that provides high sample throughput. Two cell systems will be evaluated, HepaRG cell line and primary hepatocytes. The genes associated with metabolism are known, and they will be incorporated into the assay. miRNA associated with metabolism are not known, and they will be identified using a whole transcriptome miRNA assay. The final screening assay will incorporate both mRNA and miRNA, and will be performed in a 384-well format. The data generated will be dose response data so that differential metabolism can be assessed based of what genes are regulated as well as the EC50 at which they are regulated. The significance is that this assay will enable the screening of compounds for metabolism on a cell system that mirrors in vivo liver function and assess all the pathways by which compounds are metabolized, including Phase I and Phase II metabolism, and transporters, so that not only will the primary compound effects be monitored, but also the effect of metabolites produced by the cell system will be assessed. This will be done ins a single assay, something that cannot be done with enzyme assays. Acquiring dose response data and EC50's at the level of gene expression is highly novel, and brings gene expression up to the level where metabolism enzyme assays are today.
