Discussion

1. Mouse embryonic fibroblasts from 32 different inbred mouse strains are simultaneously grown and seeded onto single 384-well plates. SNPster (haplotype association mapping) Cell permeability Cell fixation and multiplex staining after 24 h and 72 h for high-content imaging analysis NOR/LtJ WSB/EiJ Differences in dose-response curves between strains -logP Mitochondria membrane potential Hoechst Permeability dye Cytochrome c Dose: log[Cycloheximide] (nM) Control Cumulative position Cycloheximide 100mM Nonlinear regression and extraction of quantitative phenotypic data. Pharmacogenetics: In-vitro drug toxicity screening O. Suzuki1, N. Butz1, R. Singh2, B. Steffy1, D. Scoville1, B. Parks2, R. Thomas2 , T. Wiltshire1 1) School of Pharmacy, University of North Carolina, Chapel Hill, NC; 2) The Hamner Institute for Health Sciences, Research Triangle Park, NC. Introduction Pharmacogenetic studies have successfully identified genetic variants that contribute to variation in susceptibility to drug responses, but it is still a complex and challenging task to evaluate broadly across the human genome to identify the genetic components of response to drugs. Despite limitations there is a pressing need to identify genetic components that contribute to the efficacy and toxicity of drugs, across a wide spectrum of agents. We have proposed that using an alternative model population approach will provide many underlying mechanisms and pathways that are implicated in drug activity and responses. We have developed a platform from genetically well defined mouse strains which will enable us to assess effects of toxicity and efficacy of current and novel agents in drug therapies. The use of inbred mouse strains has potential advantages in screening a large number of compounds for response variations. It is a genetically stable population that allows us to quickly perform association scans using available genotype information and the large amounts of phenotypic data generated by this project. Here, we present a new strategy to identify genes and gene pathways that underlie susceptibility to cellular-level adverse drug reaction. No targets have been validated yet, but we demonstrate the ability to multiplex cell-based assays for high-throughput QTL discovery. Results We currently have 35 drugs screened that are in different stages of the image acquisition and analysis. We have not advanced to the point of QTL mapping yet. Partial results show some differences between strains in drug response (Figures 1 and 2). The cells are treated using 9 different drug concentrations, ranging from 15 nM to 100 mM. Figure 1: Strains display different sensitivities to Cycloheximide after 24 h of treatment. Segmentation of acquired images and calculation of different parameters relevant to cytotoxic response: Some strains display markedly different responses to drug treatment A) Cytochrome c localization and release from mitochondria Cell loss DNA content Cell membrane permeability Nuclear morphology Mitochondrial membrane potential changes B) Figure 2: Cell permeability dose-response curves (A) after 24h of treatment with Cycloheximide, showing differences between two inbred mouse strains. WSB/EiJ cells have increased cell permeability (B) after treatment with 100mM Cycloheximide for 24 h (green fluorescence). Discussion Our current results suggest that it is possible to identify susceptibility regions using this methodology. The presented method takes advantage of the large amount of phenotype data generated by high-content imaging and the detailed genotype information available for inbred mouse strains. This allows us to perform a high-throughput scan for QTLs affecting cytotoxic responses. This information, combined with the increasing amount of protein interaction and functional data available, can potentially help the discovery of genes involved in drug adverse reactions. Genome-wide association scans are used to discover QTLs involved in cytotoxic response. Methods We have screened embryonic fibroblasts from 32 inbred mouse strains with 35 toxicants/drugs in a high-content imaging screen that determines changes in specific cell-health status phenotypes (nuclear changes, membrane permeability, mitochondrial membrane potential and apoptosis). The phenotype data generated will be used in haplotype-based genome-wide association scans. The experimental design is outlined in the following sequence. Candidate gene selection based on functional and expression information and validation.