Development of Species-Specific Primary Hepatocyte Nuclear Translocation Assays for High-Content Imaging
Chad Deisenroth and Valerie Soldatow. ScitoVation.
Liver nuclear receptors play roles in xenobiotic and endobiotic metabolism, cell growth, differentiation, altered lipid metabolism and rodent carcinogenesis. Some ToxCast™ assays evaluate ligand binding to these nuclear receptors in cell‐free or cancer cell-based systems, providing questionable relevance to observed bioactivity. This project augments other efforts assessing steatosis and cell proliferation (Projects TP-IVc, d) caused by commercially important compounds that target NR pathways. In this work, we are developing an assay that measures receptor activation and translocation from cytosol to nucleus. For several of these receptors (e.g., CAR, PXR, AHR), ligand‐dependent activation results in cytoplasmic‐to‐nucleus translocation, a step that is followed by target gene transactivation. As an early molecular initiating event to nuclear receptor‐induced gene programs, visualization and detection of nuclear shuttling provides key information for mode of action (MOA) determinations. In addition, primary hepatocyte models provide a phenotypically relevant environment for reproducing the cellular regulatory networks that regulate nuclear translocation. The objective for this project is to develop a cross-species, hepatocyte based platform to investigate xenobiotic-induced nuclear receptor shuttling using a viral delivery system for nuclear receptor‐fluorescent fusion proteins.
In this project, we have completed the genetic engineering of adenovirus expression constructs for human, mouse, and rat Pregnane X Receptor (PXR)-Red Fluorescent Protein (RFP) reporters. Application of functional adenovirus particles for the respective PXR constructs has been initiated in rat hepatocytes, with subsequent development of mouse and human hepatocyte systems to ensue later. A high-content imaging analysis algorithm is currently in development to track reporter localization using model PXR reference activators in the rat system. Method development will be followed by training set validation with an established set of chemical and drug compounds with known activity for species-specific PXR receptors.
Implications: The progression of toxicology research from observation of apical endpoints in rodent in vivo studies to MOA elucidation at a cellular and molecular level has transformed the way we think about chemical perturbations. A transition to in vitro assessments provides an opportunity to capitalize on the knowledge afforded by molecular toxicology to address key events occurring across the MOA spectrum: from nuclear receptor activation to gene expression to phenotypic outcomes. Establishment of a translocation assay for key nuclear receptors in primary hepatocytes will provide a valuable fit-for-purpose tool for chemical screening, prioritization, and MOA determinations. Coupled with other “omics” technologies and phenotypically relevant assays, HCI- translocation assays provide a key component to developing a full suite of assays for in vitro based risk assessments.
Key words: primary hepatocytes, high content imaging, nuclear receptors
Project start and end dates: January 2015 – December 2016
Peer-reviewed publication(s): None to date
Abstract revision date: February 2016
This abstract was prepared by the principal investigator for the project. Please see www.americanchemistry.com/lri for more information about the LRI.