Biliary and Intestinal Cell Models of Drug Toxicity

描述（由申请人提供）：

DESCRIPTION (provided by applicant): Recent, unprecedented expansion of candidate drugs has generated a critical need for early phase toxicity testing by in vitro assays that are rapid, sensitive, reproducible and cost effective There is a particular need for cytotoxicity assays that monitor injury to cholangiocytes, epithelial cells of bile ducts (BDC), or to enterocytes, epithelial cells of the small intestine (IEC). Although these sites are known targets of widely used anticancer drugs, in vitro assays have not been established due to the lack of appropriate cell systems The goal of the proposed research is to develop cytotoxicity assays using newly available cell systems and bile from drug-treated animals as a physiologically relevant way to expose the cells to reactive drug/metabolites, which is the most plausible route of exposure. The anticancer drugs [Iomustine (CCNU), methotrexate (MTX), docetaxel (DCT)] chosen to validate these in vitro cytotoxicity models have well-established toxicities to BDC or IEC in vivo, represent classes of drugs that work by different mechanisms, and are extensively excreted in bile. Our approach is based on preliminary studies indicating that bile from rats treated with the cholangiodestructive agent 4,4'-diaminodiphenylmethane (DAPM) is cytotoxic to primary rat BDC in vitro. Functional and morphological characteristics of DAPM-induced injury to bile ducts in vivo are virtually identical to the BDC injury produced in vitro. Bile from rats treated with the NSAID indomethacin has been reported to injure a rat intestinal cell line in vitro. A more differentiated cell line that forms villus-like enterocytes (Cdx-IEC-6 cells) is proposed as the model IEC system in this application. Our specific aims are (1) validate the target organ specificity of biliary drug/ metabolites to BDC versus IEC in vitro by viability assays and functional indices of cell injury, (2) characterize the specificity, reproducibility and high-throughput possibilities of sensitive assays for mitochondrial injury, paracellular permeability, ATP content, and membrane leakage as potential biomarkers of biliary drug/ metabolite-induced dysfunction, and (3) determine if biliary drug/metabolites induce comparable early effects on gene expression in BDC and IEC in vivo versus in vitro using laser capture microdissection and gene chip analyses. The basic techniques developed in this research will lead to new methods for screening potential drug injury to target sites that are not easily investigated in vivo.