Office of Biological and Environmental Research

DOE Low Dose Radiation Program Workshop V

2005 Abstract

Title: Using a Low LET Electron Microbeam to Investigate Non-Targeted Effects of Low Dose Radiation

Authors: William F. Morgan¹ and Marianne B. Sowa²

Institutions: ¹Radiation Oncology Research Laboratory, University of Maryland, Baltimore, Maryland; ²Chemical Structure and Dynamics, Pacific Northwest National Laboratory, Richland Washington

We have recently installed a low-linear energy transfer (LET) electron microbeam that generates energetic electrons to mimic radiation damage from gamma- and x-ray sources. It has been designed such that high-energy electrons deposit energy in a pre-selected subset of cells, leaving neighboring cells unirradiated (Figure 1). In this way it is possible to examine non-targeted effects associated with low dose radiation exposure, including induced genomic instability and bystander effects.

Figure 1. Schematic figures of the electron microbeam (A) and the electron microbeam - sample interface(B).

Our studies take advantage of a high-throughput assay for hyper recombination and deletion events. This assay was developed during the current funding period of our DOE grant and utilizes a novel green fluorescence gene (GFP) reporter assay. A summary of the experimental strategy for determining delayed radiation-induced genomic instability is presented in Figure 2. Analysis of stable (GFP- or GFP+) colonies and unstable (GFP+/-) colonies has been fully automated using fluorescence microscopy and provides a robust, reliable, highly sensitive assay for detecting delayed events occurring in cells exposed to low doses of ionizing radiation.

Figure 2. Protocol for evaluating induced genomic instability using the GFP reporter assay in RK036 cells. RK036 cells contain a mixture of GFP+ (green) and GFP-(clear) cells. They will be plated at low density for colony formation and instability evaluated using our automated flourescence microscope. A colony will be considered stable if all cells are green or all cells are clear, and unstable if there is a mixture of GFP+ and GFP- cells in the colony. Our usual criteria for instability is >5 cells of one cell type (e.g., GFP+) cells in a colony containing predominantly GFP-cells.

We will present the data on induced genomic instability using the GFP reporter assay and on bystander effects from our low dose/low dose-rate experiments using x-rays and preliminary results using the low-LET electron microbeam.

This work was sponsored by the Biological and Environmental Research Program (BER) U.S. Department of Energy (to MSR); Grant number: DE-AC06-76RLO; Grant sponsor: U.S. Department of Energy (to WFM); Grant number: DEFG02-01ER63230.

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