High throughput assays of Bone Marrow Genotoxicity After Radiation Exposure

University Of Rochester, Rochester NY

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Abstract

In instances of accidental radiation exposure or radiological terror events, incomplete or missing physical dosimetry will be the norm. EPT dosimetry of tooth or bone (Project 3) is potentially viable, but does not address particulate or neutron exposures. Biodosimetry (Bd) is therefore needed to make rational clinical management decisions, and for long-term carcinogenic risk assessments. Currently there are no feasible means for evaluating the cytogenetic effects of radiation to bone marrow. Traditional Bd techniques (dicentrics and micronucleated lymphocytes) predominately measure effects to cells in peripheral circulation. These assays are also too cumbersome to monitor large populations. The goal of this project is to supply the Nation with high throughput radiation research and Bd tools that overcome these limitations. The assays that will be optimized and validated are 1) a flow cytometry (FCM) based method that provides for rapid acquisition of in vivo bone marrow genotoxicity data, and 2) a three dimensional (3-D) human bone marrow culture system that represents a powerful ex vivo investigative model. These systems will be useful for numerous investigations into radiation-induced cytogenetic damage. In regard to the current grant application, they will be important for assessing the ameliorating effects of experimental radioprotective agents. Furthermore, these complementary in vivo and ex vivo assays may represent a rapid bone marrow Bd tool. That is, in conjunction with radiation response calibration curves generated from the ex vivo 3-D bone marrow culture, the in vivo assay may enable acquisition of timely bone marrow genotoxicity and dosimetric information in large-scale radiological emergency situations. The endpoint that we will utilize as a chromosome damage indicator is micronucleus formation in immature reticulcoytes (MN-RET). Advantages of MN-RET as a radiation cytogenetic damage endpoint include the following: 1. compatible with rapid FCM analysis, 2. minimal sample processing required, does not require cell culture, 3. provides a sensitive measurement of cytogenetic damage to an otherwise inaccessible tissue - bone marrow, and 4. reticulocyte-based assays are already FDA-approved for testing drugs - radiation damage is a natural extension.