Superfund Research Program
Image Analysis and Bioassays Core
Project Leader: Robert C. Burghardt
Grant Number: P42ES004917
Funding Period: 2000-2008
The Image Analysis and Bioassays Core worked closely with SBRP investigators to provide routine and novel analytical microscopy and image analysis support services. Improvement in analytical microscopy instrumentation and services is an ongoing priority and new facilities added during the past year included: (1) a live cell imaging workstation supported by an Olympus CK30F inverted microscope interfaced with a Hamamatsu ORCA ER high resolution digital CCD camera, motorized stage, Compix and Metamorph image acquisition and analysis software, (2) a Leica EM Uc6r ultramicrotome and, (3) Leica EM TP automated tissue processor. New applications were added to address experimental needs of SBRP investigators who are utilizing vital imaging capabilities of multiphoton microscopy instrumentation. Projects 1-3 and 6 made extensive use of vital imaging tools and applications for routine bioassays of cellular function in toxicant-exposed cell types including: the fluorescence recovery after photobleaching (FRAP) approach to quantify effects of PAH mixtures on intercellular communication (a tumor promoter assay) and assessment of cytochrome P450 enzyme induction (EROD assay); single-cell and bulk analysis of cytochrome P450 enzyme induction; and analysis of intracellular Ca2+ homeostasis and signal transduction. New methods were added to exploit the inherent fluorescence properties of PAHs to analyze uptake and partitioning of these compounds in exposed tissues as well as to relate cell and tissue distribution to alterations in cellular homeostasis. Precision cut-tissue slices are being combined with non-invasive imaging tools in an effort to develop novel risk assessment strategies designed to bridge the gap between in vitro and in vivo models for mechanistic analysis of cellular injury. Project 1 also utilized immunocytochemistry as well as direct analysis of estrogen receptor (ER), aryl hydrocarbon (AhR), Sp- family member, and coactivator protein-protein interactions using fluorescence constructs in conjunction with a fluorescence resonance energy transfer (FRET) assay. Improvements in FRET analysis (precision FRET) were introduced to permit quantitative kinetic analysis of the extent and duration of these protein-protein interactions. Projects 2 and 3 utilized fluorescence bioassays for the assessment of genotoxicity or cytotoxicity of PAH mixtures and heavy metals on liver cells or mesangial and glioma cells, respectively. The Core also provided cell culture support and training along with assessment of functional endpoints in cells (EROD and FRAP assays) to support risk assessment studies of complex mixtures required in Project 6.