Funded Pilot Projects

  1. Standards for L-band Tooth Dosimetry [back to top]
    Sandy Eaton, Ph.D. [PI]
    Department of Chemistry and Biochemistry
    University of Denver,Denver

    Designated Liaison at Dart-Dose CMCR: Hal Swartz

    Abstract: One of the goals of the Dart-Dose CMCR is focused on the development of a field deployable physical biodosimetry utilizing EPR measurements of teeth. Differences between mouths of individuals mean that it is very important to have a standard reference sample built into the dosimeter to provide a calibration of each measurement. We propose to prepare and test materials that could be more effective as a standard than the one that is currently used in the prototype dosimeter.

    In the first year of the project we identified absorbing polymers in polymer tubing as promising bases for 15N-PDT standard samples. During the second year of funding,the following will be done:

    • Test water absorbing polymers.
    • Work with Dart-Dose CMCR staff on the geometry for the standard sample and total spins required for that geometry.
    • Develop a protocol for reproducibly loading the PDT onto the selected absorber and reproducibly filling the tubing.
    • Engineer the sample container including testing various closure systems.
    • Test the chemical stability in the polymer environment. We do not expect the nitroxide to react with the polymers, but this needs to be checked.

    Relevance: The goal of this project is to provide a well-defined reference standard for use in the field deployable physical biodosimetry for EPR measurements of teeth that is under development by Dart-Dose CMCR. This standard will improve the precision of the measurements, which will permit greater confidence in determination of the received dose.

  2. Reference Materials for Development and Deployment of EPR Biodosimetry Systems[back to top]
    Drago Skrtic, Ph.D. [PI]
    Paffenbarger Research Center, ADA Foundation (located on the NIST campus)
    Marc Desrosiers, Ph.D.
    National Institute of Standards and Technology (NIST), Washington, DC

    Designated Liaison at Dart-Dose CMCR: Hal Swartz

    Abstract: The project will develop an enamel-equivalent EPR reference material to aid in the research and deployment of EPR-spectrometer-based retrospective biodosimetry systems. This reference material will be synthetic carbonated hydroxyapatite (CHA) that should possess sufficient uniformity and reproducibility suitable for mass-production scalability. Gamma-ray irradiation from NIST-calibrated Co-60 sources will be used to control the EPR characteristics of the standard. This reference material will be an essential tool in basic research and a key component of a field-deployable EPR (FD-EPR) biodosimetry system.

    Specific Aims for the Second Year:

    1. The batch of HAC powdered reference material will be prepared using the procedure developed in the first year of the project.Powdered HAC mixed with polyethylene as a binder will be fabricated into small pellets, gamma irradiated and tested for physical and chemical stability, uniformity and EPR spectra stability (X-band EPR spectrometery).
    2. The mixture of HAC and polyethylene will be prepared in the shape that mimics the geometry of an incisor tooth so that it can be placed in the same position in the EPR instruments as an isolated real tooth. The EPR measurements(L-Band EPR Spectrometry) of the material in the shape of incisor tooth will be performed at Dartmouth to relate the properties to what will be observable in the appropriated instrumental configuration.The physical, chemical and EPR stability of the reference material prepared in the shape of incisor tooth will be tested.

    Relevance:Tooth enamel as reference tool is not ideal due to its variable physical composition and/or unknown radiation history.Our results from the first year of this project showed that synthesized enamel-like HAC powder comprised EPR characteristics that are equivalent to that of irradiated human enamel.This HAC as a reference material could ensure consistent testing across the entire Dart-Dose CMCR research project.Moreover, the HAC can be fabricated into a variety of shapes with adjustable EPR signal levels. Ultimately this HAC reference material can be used for QA/QC assessments of the FD-EPR manufacturing process,as well as a field standard for FD-EPR setup, and serve as a field measurement normalization standard.The synthetic tooth-like standard will support research and development across a broad range of present and future activities of the CMCR and partners. CMCR basic R&D is performed in several locations, both local and remote;the standard will permit real-time direct comparisons of hardware/software capabilities and modifications.

  3. Wireless microsensors for EPR dosimetry [back to top]
    Barjor Gimi, Ph.D. [PI]
    The Geisel School of Medicine at Dartmouth and Thayer School of Engineering

    Designated Liaisons at Dart-Dose CMCR: Ben Williams and Paul Meaney

    Abstract: The aim of the proposed project is to devise self-resonant radio frequency (RF) detectors or EPR tooth dosimetry. Microelectromechanical systems (MEMS) fabrication techniques will enable low cost and high volume production. These detectors will be designed to reversibly adhere to tooth surfaces and inductively couple with an RF coil on a hand-held EPR probe, thereby possibly reducing the incidence of breakage of the hand held probe which in turn would save valuable measurement time. The detector geometry will be optimized to obtain signal from the enamel and therefore to minimize the contributions of extraneous signal to dosimetry measurements. Detection sensitivity and inter-detector variability will be quantified.

    Report and Aims for Second Year: In the first year of our pilot project,we successfully designed, fabricated and tested self-resonant microresonators intended for wireless EPR tooth dosimetry. We demonstrated that the microresonators could be fabricated in a low-cost, high throughput manner that is conducive to widespread use required in the event of a large population being exposed to a radiation accident. Based on lessons learned from our initial experiences, we propose to develop larger resonators for more effective inductive coupling with the external loop resonator currently available at Dartmouth for EPR dosimetry. The EPR group at Dartmouth (Dart-Dose CMCR) intends to use numerical simulations to explore a variety of geometries to determine the ones that may be most suitable for both effective coupling with the external resonator and for extracting signal principally from tooth enamel. Here, if a single-plane resonator geometry is determined to be conducive to efficient EPR tooth dosimetry, we shall devise fabrication methods on flexible substrates so that these resonators may be conformally attached to tooth surfaces.

  4. Role of endotoxin antagonists as novel radiation mitigation agents [back to top]
    Ofer Levy, M.D. [Co-PI]
    Children's Hospital Boston and Harvard Medical School
    Lee Nadler, M.D. [Co-PI]
    Dana-Farber Cancer Institute; Harvard Medical School

    Designated Liaisons at Dart-Dose CMCR: Hal Swartz and Eva Guinan

    Abstract: Radiation exposure may occur in the context of medical diagnostics, medical therapy, or due to war or terrorism. A common feature of radiation injury is mucosal barrier injury (MBI) that weakens the gut barrier thereby allowing translocation of microbes and microbial products into the systemic circulation. We have already established in Balb/c mice that TBI in the 6-8 Gy range is associated with endotoxemia and activation of endotoxin-directed innate immunity, including altered levels of LPS-interactive proteins. We have discovered that administration of a 21 kDa recombinant fragment of the antibacterial and endotoxin-neutralizing protein recombinant bactericidal/permeability-increasing protein (rBPI21) starting 24 hours after TBI significantly reduced bone marrow toxicity. The mitigating effects of rBPI21 on TBI induced marrow injury have implications for therapy in that a growing list of endotoxin-related biomarkers are available for potential biodosimetry and multiple endotoxin and/or Toll-like receptor 4 (TLR4; endotoxin receptor) antagonists are in biopharmaceutical development. As rBPI21 has both antibiotic and endotoxin neutralizing properties, its mitigating effects may relate to either or both mechanisms of action. Eritoran (Eisai Pharmaceuticals) is a synthetic antagonist of lipid A (the bioactive core region of LPS) that potently and effectively blocks LPS-induced signaling via TLR4 without any antibacterial effect. Eritoran is readily synthesized in large amounts, is stable in lyophilized form, has demonstrated safety in human trials and potently blocks endotoxin in vitro and in vivo. We therefore propose to evaluate the radiation mitigation potential of Eritoran in comparison to rBPI21 in relation to endotoxin-related biodosimetry markers. With first year funding, in pursuit of our CMCR-funded work, we have begun to assess whether milk products including skim milk powder, rich in anti-infective components such as immunoglobulins and antimicrobial proteins/peptides , or anti-ETEC bovine colostrum [6] may provide benefit when administered 24 hours post-irradiation in conjunction with oral flouroquinolone antibiotic. Powdered skim milk powder (Carnation Skim Milk Powder; Nestle) and anti-ETEC bovine colostrum (Immuron; Melbourne, Australia) were reconstituted at a low (4mg/mouse/day) and high (40mg/mouse/day) dose in autoclaved tap water. Assuming each mouse drinks ~5mL/day, bottle dosing was estimated at 0.8mg/mL of ETEC for low dose (4mg) and 8mg/mL of ETEC for the high dose (40mg). Fresh batches of skim and ETEC solution were made every 48 hours throughout the 14 days of milk product administration. Of note, mice receiving 7Gy and no mitigation agent all died by Day 16. In contrast, mice receiving oral antibiotic showed somewhat longer time to death with 100% mortality by Day 23. In contrast, a substantial proportion (~25-40%) of mice receiving both antibiotic and milk product (either skim milk or ETEC) demonstrated long-term survival to Day 30. As an additional indicator of well-being, we also measured the impact of enrofloxacin with or without milk products on the weight of irradiated mice. In this first pilot study, combined treatment with oral antibiotic and milk products demonstrated trends towards greater weight gain. Bone marrow toxicity is an important determinant of the degree of radiation-induced morbidity and mortality. To assess whether the candidate mitigation regimens tested may impact radiation-induced marrow toxicity, bone marrow cellularity was assessed by counting trypan blue-excluding vital cells from flushed bones. This analysis revealed that the groups treated with oral antibiotic and anti-ETEC colostrum had substantially higher median BM mononuclear cells per limb, raising the possibility that anti-ETEC might enhance or speed bone marrow recovery after radiation. Overall, these pilot results demonstrate the feasibility of our model and raise the possibility that providing oral colostrum- or milk-derived products, a rich source of both caloric nutrition and anti-infective molecules, may reduce radiation-induced morbidity and mortality. Future proposed work will determine whether any effect is reproducible and, if so, characterize the underlying mechanisms with respect to impact on bacterial translocation and/or endotoxemia, as outlined below.

    Second year work proposed:

    • Aim 1: Compare the efficacy of bovine colostrum, 17R-RvD1/17R-RvD2, and DHA given alone or in combination with the flouroquinolone antibiotic enrofloxacin in enhancing survival after lethal TBI in a murine model.
    • Aim 2: Characterize the relative effects of bovine colostrum, 17R-RvD1, and/or RvD2 and DHA on bacteremia and activation of endotoxin-directed innate immune responses in relation to survival.

    Relevance: Overall, the proposed studies employ innovative approaches to assess endotoxin-directed innate immunity for biodosimetry and radiation mitigation technology. Thus, successful pursuit of these hypotheses may ultimately enable a technology that not only assists in staged biodosimetry but also links the bioassay result to a specific mitigation therapy, namely endotoxin inhibition.

  5. Compact Biodosimetry Probe based on ESR Signals from Incisor Teeth [back to top]
    Aharon Blank, Ph.D. [PI]
    Schulich Faculty of Chemistry
    Technion - Israel Institute of Technology, Haifa, Israel

    Designated Liaisons at Dart-Dose CMCR: Ben Williams and Piotr Lesniewski

    Abstract: The objective of this project is to develop a demonstration of a lab prototype which is a simple, affordable, compact and transportable tool for quick and reliable biodosimetry, based on pulsed Electron Spin Resonance (ESR) signals from incisor teeth. The probe will be designed, constructed, integrated with a pulsed microwave system, and finally tested with irradiated teeth. It will involve the use of new methods such as the measurement of ESR signals outside a permanent magnet (the so-called "ex-situ" approach), combined with miniature sensitive microwave resonators. The assembled system and the results of its experimental tests will provide first-hand evidence to the possibility of using a relatively simple and small (but nevertheless accurate and effective) pulsed ESR system and probe for biodosimetry. The final outcome will also provide information about the system's spin sensitivity, which is a direct measure of its radiation-dose sensitivity and accuracy.

    This proposal is aimed at the development of a prototype of a small hand-held probe and a system for fast and accurate measurement of radiation absorbed dose in humans, based only on signals imprinted in the teeth. Such measurements are important for the diagnosis of patients arriving to hospitals following an unexpected nuclear event, such as a terrorist attack or a nuclear power plant accident.

    The objective of this project is to develop a demonstrative laboratory prototype of a simple, affordable, compact and transportable tool for quick and reliable biodosimetry, based on pulsed ESR signals from incisor teeth.The probe will be designed, constructed, integrated with our pulsed microwave system, and finally tested with irradiated teeth. This will provide first-hand experimental evidence of the possibility of using a relatively simple and small (but nevertheless accurate and effective) pulsed ESR system and probe for biodosimetry. The final outcome will also provide information about the system's spin sensitivity, which is a direct measure of its radiation-dose sensitivity and accuracy. If successful, this demonstrative pilot project can be later followed by a more practical effort aimed at bringing such type of tools to many potential users.

    To summarize: The project's progress follows closely the original work plan and timetable. Our probe's simulated performance follows closely the actual measured data in terms of static field, microwave field, and resonator, all the way to the predicted signal from irradiated teeth (using an estimated 6.3x1013 spins per gray per gram3). The concept of an ex-situ compact pulsed ESR probe for biodosimetry using incisor teeth has shown its feasibility, and we are currently completing some final tests and gathering the data required to produce a manuscript that will provide all relevant details of the probe, our pulsed system, and the experimental results.

  6. Influence of commercial resins on the dosimetric signal recorded in teeth [back to top]
    Bernard Gallez, Ph.D. [PI]
    Medicinal Chemistry and Radiopharmacy
    Université catholique de Louvain, Brussels, Belgium

    Designated Liaisons at Dart-Dose CMCR: Ben Williams and Ruhong Dong

    Abstract: In vivo electron paramagnetic resonance spectroscopy is a powerful method to quantify concentrations of radiation-induced radicals within intact teeth. In tooth enamel, the irradiation creates carbonate radical centers within the hydroxyapatite matrix in proportion to the absorbed dose. These free radicals remain stable for a long period of time and can be detected in vivo using low frequency EPR. The individual determination of tooth EPR signal intensity can thus lead to a powerful dose estimation for a preliminary triage of suspected irradiated people. In the context of the present application, we will focus on the possible interference coming from restorative materials, specifically the dental resins. The use of synthetic resins is increasingly popular in routine dentistry practice. Dental resins are polymerized in situ using visible light to give a very hard material. Interestingly, this material is almost not visible to untrained individuals. It has been established that tooth restoration may lead to the formation of stable free radicals easily evidenced by EPR spectrometry. These radicals are relatively stable, and it may take months before disappearance. After irradiation, some resins may also present some radiation-induced signals. If present, an EPR signal coming from the resins, which are virtually invisible for untrained people, could possibly interfere with the classical dosimetric signal. It is therefore crucial to get more knowledge on this possible confounding EPR signal. The overall aim of this application is to study the influence of the dental resins on the dosimetric signal recorded in irradiated teeth. Six specific aims will be investigated:

    1. To screen the EPR signal generated during the polymerization of dental resins;
    2. To study the kinetics of decay over the time of the EPR signal coming from these materials;
    3. To study the influence of irradiation on old resins after the fading of the EPR signal originally coming from the photolymerization;
    4. To study the influence of the restoration on the EPR dosimetric signal recorded in teeth by a systematic study comparing non-restored teeth and restored teeth before and after irradiation;
    5. To study the influence of the previously described factors using up-to-date dosimetric systems developed in CMCR-Dartmouth;
    6. To provide a rationale to include or exclude teeth that were restored, and to propose possible clues to eliminate this possible confounding effect.

    Relevance: EPR tooth dosimetry is a particularly attractive approach for initial triage, as well as an option for use in dose estimation to guide subsequent treatment. To achieve the goal of an EPR-based efficient triage, it is essential to make sure that the EPR signal recorded is directly linked to the dosimetric signal, and not linked to a possible confounding factor. In the present proposal, we plan to focus on the possible contribution of dental resins to the signal recorded in irradiated teeth.

    Preliminary Findings:

    Screening of the EPR signal generated during the photopolymerization of dental resins.

    Twenty commercially available composites on the US market were selected because they are widely used for restoration of incisors and canines. Small size samples (30-40mg), corresponding to a medium-size restoration for anterior teeth, were light cured and measured in X- and L-band. The normalized signal measured shortly after polymerization in the resins ranged from 6.0±0.2 to 38.3±1.1 (X-band). Compared to the radio-induced signal in dental enamel irradiated at 3 Gy (I= 0.3), the observed initial intensity was ~20 to more than 100 times higher.

    Kinetics of decay of the signal was followed by repeated measurements according to the following scheme:

    • once a day during the first week post light curing
    • twice a week during the first month
    • twice a month during the following months

    The observed kinetics was compatible with a bi-exponential decay, characterized by an early phase with a very fast decay (ranging from 1.5h for N'Durance to ~82h for Grandioso) with a median T1/2 of 16.3h. The second phase of the decay is much slower with a median T1/2 of 320h.

    Overall, the signal completely disappeared (X-Band) in 12 out of 20 resins after a maximum of 5 months after polymerization. Measurements are still on-going for 4 composites. In 3 composites,(IPS Empress, Tetric EvoCeram, Grandioso), an atypical residual signal was still observed after 5 months. It did not show any further evolution with time. The shape of this signal was different from the classical 9-line spectrum. The exact influence of this signal on the RIS remains to be appraised.

  7. Thermal Tomography as a predictor for Radiation Induced Skin Injuries.[back to top]
    James C.H.Chu, Ph.D. [PI]
    Katherine L. Griem, M.D
    Radiation Oncology Department
    Rush University Medical Center,Chicago

    Designated Liaisons at Dart-Dose CMCR: Hal Swartz

    Abstract: During a massive radiation disaster, such as a nuclear power plant incident or a "dirty bomb" terrorist attack, a large number of patients who are or are suspected to be exposed will come to hospitals to seek evaluation or treatment. It is therefore crucial to have evaluation technologies that can rapidly identify the individuals who are at risk of developing acute radiation injury requiring clinical treatment.

    The broad long-term objective of this proposal is to develop a non-invasive infrared-based imaging method that can be quickly deployed in the field to screen the skin's thermal properties for a large patient population after a radiological/nuclear mass exposure event. Skin is an ideal biomarker for this application for the following reasons: it is always in place, its radiation induced-effects have been studied extensively, it provides information related to partial body exposures, it covers the entire body, early radiation-induced skin reactions are observed within a few hours, and it plays an important role in radiation-induced multi-organ injuries/failures. 3D thermal tomography (3DTT) is a new image-processing method that can construct cross-sectional images based on the heat conduction properties of tissues. This imaging method has been a preferred technology for non-destructive evaluation of advanced ceramic components. For example, it was selected the primary tool to inspect the space shuttles after the Columbia accident. 3DTT has great potential to detect radiation-induced skin changes before the clinical symptoms develop.

    Our preliminary studies show excellent correlation between 3DTT data and radiation-induced skin reactions at 40Gy. We propose to further test this idea on mice and on human subjects. Our specific aims are 1) to determine relationships between radiation-induced skin reactions and changes in tissue thermal effusivity in mice over 2-20 Gy range, and 2) to determine measurement accuracy for skin thermal effusivity in human subjects by studying the effects from potential confounding environmental factors. The study will open a new area of research as knowledge on tissue thermal property changes in irradiated tissues have not yet been studied.

    Relevance: This proposed study will provide not only an objective method of quantifying radiation-induced skin reactions but also opportunities to offer more effective patient-specific treatment strategies during mass radiation exposure events. This proposal is highly relevant to the mission of NIH as it pursues fundamental knowledge of the nature (thermal property of tissue) and applies it to improve the treatment of human injuries.

  8. Radiation Dosimetry Using Time-domain(Pulsed)EPR Spectroscopy[back to top]
    David Gladstone, Sc.D. [PI]
    Division Of Radiation Oncology
    Dartmouth-Hitchcock Medical Center
    The Geisel School of Medicine at Dartmouth College
    Sankaran Subramanian, Ph.D.
    Pulsed EPR Laboratory
    National Cancer Institute, NIH

    Designated Liaison at Dart-Dose CMCR: Ben Williams

    Abstract: There is considerable interest in retrospective dosimetry in view of possible terrorist attacks with radioactive materials, as well as nuclear reactors going out of control either due to the failure of control mechanisms or due to natural disasters such as earthquakes and tsunamis occurring near nuclear reactors. In order to stratify people into appropriate treatment arms following such events, it is important to have a quantitative method with a large throughput to measure the dose of radiation received by an individual following adverse nuclear events.

    Electron Paramagnetic Resonance (EPR) spectroscopy is perhaps the only direct method to quantitatively assess dose retrospectively, by measuring the concentration of radiation induced radicals, e.g. in tooth enamel. These radicals are stable for thousands of years and are therefore suitable, with calibration against known doses, to determine dose at any time after exposure. While advances have been made to use continuous wave EPR for retrospective dosimetry, a pulsed Fourier transform (FT) approach has not been tried. This proposal will investigate using FT EPR for dosimetry. The FT approach has the potential to improve the sensitivity of the measurements by overcoming problems such as a weak signal of carbon radicals and the low signal-to-noise of the spectra.

    Relevance: An alternative method for conducting EPR tooth dosimetry would be an important advance if it could help improve the sensitivity of the method.

  9. PCC in Mass Casualty: Rapid Dose Assessment & Detecting Heterogeneity of Exposure[back to top]
    Firouz Darroudi, M.D., Ph.D. [PI]
    Department of Toxicogenetics
    Leiden University Medical Center, The Netherlands

    Designated Liaison at Dart-Dose CMCR: Hal Swartz

    Abstract:In case of radiation accident and specially in mass casualty scenarios two vital information need to be determined are: First, the absorbed dose, as quick as possible (within few hours), and second, namely for high doses of exposure to determine the fraction of body exposed. The first issue will give the possibility to separate people into for example two or more groups having received low, medium and high doses, consequently to assess the threshold limit for significant risk in radiation accident victims. PCC technique in which interphase cells are fused with Chinese hamster ovary mitotic cells using polyethylene glycol leads to a rapid prophasing-like reaction (after just one hour incubation), the nuclear envelope assembles and the chromatin condenses into chromosomes. The PCC technique therefore enables direct observation of radiation-induced chromosome damage in un-stimulated human peripheral blood lymphocytes. This issue is very important because dose assessment can be done within 3 hours after receiving blood samples. It is planned to establish a calibration curve for X-rays (low and high doses), and to study radiosensitivity of different individuals. Currently, one of the challenges for biological based biodosimetry assays in that induced frequency may decrease with post-exposure time (except translocation). Therefore, attempts will be made to investigate the influence of post-exposure time (from 1 hour up to 7 days) on the frequency of PCC. Taking into account that unlike other biological assays such as dicentrics and micronuclei at high doses (>3 Gy) mitotic delay and cell death have no influence on PCC, attempts will be made to explore the potential of PCC technique for discriminating between whole and partial body exposure in a simulated mass casualty accident. Peripheral blood lymphocytes from different donors will be irradiated with different doses of X-rays (2 to 8 Gy), and will be mixed with different proportion with unirradiated lymphocytes of the same donor, the Poisson or over-dispersed distribution of PCC will be used to elucidate the irradiated / unirradiated fractions. The outcomes can set standards to determine dose of exposure within just few hours after collecting blood, furthermore, to determine heterogeneity/homogeneity of exposure to design most suitable therapy regimen.

    Relevance: In order to set up appropriate standards to deal with public health and safety in scenarios of terrorist attacks and mass casualties following exposure to ionizing radiation, it is vital to determine the dose- and heterogeneity-of exposure in a shortest period of time. The proposed biological model (PCC) and designed experiments have unique advantages that can shed light on all these issues.

  10. Investigation of Dose Formation in Nails Under Fallout Radiation[back to top]
    Valeriy Skvortsov, Ph.D. [PI]
    Alexander Ivanniknov, Ph.D.
    Artem Khailov, Ph.D.
    Laboratory of Experimental Nuclear Medicine
    Medical Radiological Research Center, Obninsk, Russia

    Designated Liaison at Dart-Dose CMCR: Ben Williams

    Abstract: The aim of the project is to determine conversion coefficients between the dose absorbed in human nails and the whole body dose in conditions of irradiation at radioactively contaminated territory. It will be performed by calculations using the stochastic simulation of ionizing particles transport (Monte Carlo method) for the human phantom at different geometry of irradiation. Contribution to dose in nails caused by different types of irradiation, such as photons, neutrons and beta radiation at different energies will be considered. Dose in nails at irradiation by photons is expected to be different from the whole body dose because the nails are placed on the surface and irradiated in conditions of lack of secondary electron equilibrium. Calculations will be verified by experiments with irradiation of the nail equivalent material in phantom. Conversion coefficients will be determined for conditions of energy spectrum and geometry of irradiation similar to that at radioactively contaminated territory. Conversion coefficients are necessary for assessment of the whole body dose received by a victim of an overexposure from fallout radiation using absorbed doses in nails, which may be accessed by electron paramagnetic resonance (EPR) spectroscopy method.

    Relevance: This project has obvious relevance for Dart-Dose CMCR's in vivo nails project as well as implications for the in vitro(clipped) nails project. It also could be important for assessing whole body versus partial body exposure in terrorist events involving radiation.

  11. Intestinal Microbiota for Radiation Biodosimetry[back to top]
    John Baker, Ph.D. [PI]
    Department of Surgery
    The Medical College of Wisconsin

    Designated Liaison at Dart-Dose CMCR: Eva Guinan

    Abstract: The primary purpose of this Pilot Project in Biodosimetry is to continue our research and development on intestinal microbiota as novel biomarkers of prior radiation exposure. There is an urgent need for rapid, accurate and sensitive diagnostic platforms to confirm exposure to radiation and estimate the dose absorbed by individuals subjected to acts of radiological terrorism or nuclear power plant accidents. Clinical symptoms and currently available dosimeters, even when available, do not provide adequate diagnostic information to triage and treat life-threatening radiation injuries. We have identified intestinal microbiota as novel biomarkers of prior radiation exposure.

    In preliminary studies male Wistar rats received single fraction total body irradiation of 10 Gy. Fresh fecal pellets were obtained from each rat prior to (day 0) and at days 4, 11 and 21 post irradiation. Fecal microbiota abundance was determined using microarray analysis and quantitative PCR. The sequence of the 16S ribosomal RNA (rRNA) gene is unique to each eubacteria species and the abundance of each genomic 16S rRNA gene is indicative of the abundance of the species. The current candidate radiation exposure biomarkers consist of sustained increases in 16S rRNA levels of 12 members of the Bacteroidales, Lactobacillaceae and Streptococcaceae after radiation exposure, unchanged levels of 98 Clostridiaceae and eptostreptococcaceae, and decreases in levels of 47 separate Clostridiaceae members. These biomarkers are found both in rat feces and in human feces.

    The extent of radiation exposure is determined from fecal samples using common instrumentation platforms operable under field-deployed conditions. There are major advantages to using intestinal microbiota as biomarkers of radiation exposure: 1) this biodosimetry technique is non-invasive; 2) microbiota provide a sustained level of reporting signals that are increased by several orders of magnitude following exposure to radiation; and 3) intestinal microbiota unaffected by radiation serve as internal controls. The biomarkers may also be indicative of early gastrointestinal system injury following therapeutic radiation. In year 1, one specific aim is proposed to further research the ability of intestinal microbiota to detect previous exposure to radiation under non-GLP conditions.

    We propose the following studies be performed:
    Determine the lowest dose of radiation detectable and earliest response by intestinal microbiota.
    Wistar rats (n=48) will be irradiated with a single exposure to X-rays over the range 1-10 Gy to determine the earliest response time and the duration of effect (1-14 days). Feces will be collected daily and analyzed with a microarray analysis signature specific for radiation exposure that we have developed. We will confirm our findings using qPCR. Success will be found by defining the dose- and time-response characteristics for intestinal microbiota to detect prior exposure to ionizing radiation. In year 2 a second specific aim will be proposed to determine the impact of the subjects' health status on intestinal microbiota after irradiation.

    Relevance: Our approach may complement the EPR studies being conducted at the Dartmouth Biodosimetry Center for Medical Countermeasure Against Radiation and thus directly address the overall goals for biodosimetry.

  12. Improving Accuracy of Automated Detection of Radiation-Derived Damage[back to top]
    Peter Rogan, Ph.D. [PI]
    Jagath Samarabandu, Ph.D.
    Joan Knoll, Ph.D.
    Department of Biochemistry
    The University of Western Ontario, London, ON Canada

    Designated Liaison at Dart-Dose CMCR: Hal Swartz

    Abstract:Biological dosimetry is an essential tool for determining the radiation dose received by an individual when physical dosimetry is unavailable, as would be the case in a mass casualty incident involving radiological or nuclear material. Ionizing radiation causes the formation of the dicentric chromosomes (DC). The dicentric chromosome assay (DCA) is currently the gold standard for biological dosimetry, as it is specific and sensitive for calibrated doses of ionizing radiation. Unfortunatley, the current methods are labor intensive,time consuming and not feasible for assessment of many individuals.The project objectives is to automate detection of DCs.The University of Western Ontario(UWO) has developed,published and validated image processing algorithms to automatically detect key chromosome features in the DCA. In this process, we determine centromere location(s) and count the number of centeromeres on each chromosome,but perform all steps in an automated fashion.During the past year,the investigators integrated these algorithms, and rewrote and tested Automated Dicentric Chromosome Identificatiom software (ADCI) in C++,which was distributed to end users and evaluated by them.ADCI is extremely fast and its parallelization for high performance cluster computing shows enormous capacity to address the processing requirements of a mass casualty radiation event.However it still has unacceptably elevated false positive detection rate of dicentric chromosomes.In this proposal,we will incorporate our newly published methods for intensity-integratd Laplacian detection of centromeres that are capable of handling a wider range of variation in chromosome morphology,including sisiter chromatid separation typically present in chromosomes prepared for biodosimetry testing.The investigators will develop supervised learning algorithms to improve DC detection accuracy.Improved timeliness of biodosimetry without loss of accuracy will result in more appropriate medical intervention and better therapeutic outcomes.The project will begin to scale development of algorithms and image processing software through support of graduate students in Pathology,Biochemistry,Computer Science and Engineering, and a computer software developer.

    The major objective of the second year is to incorporate a novel, published segmentation method which promises to significantly improve the accuracy of centromere detection in these cases, without incresing the time needed to process these samples. The secondary objective is to incorporate suggested improvements preprocessing and the graphocal user interface that will improve the usablity of the software and confirm to best practices of biodosimetery laboratories.

    Relevance: This project will provide critical data needed for assessment of overexposures to ionizing radiation using a well-validated biological dose measurement.The objective is to automated and accelerate procedures that are currently being performed manually in most laboratories without loss of sensitivity or specificity.

  13. Aspartic/Glutamic Acid Decarboxylation Dosimtery[back to top]
    Jeffrey J.Hayes, Ph.D. [PI]
    Department of Biochemistry and Biophysics
    School of Medicine and Dentistry
    University of Rochester, Rochester, NY

    Designated Liaison at Dart-Dose CMCR: Steven G. Swarts, Ph.D

    Abstract: Accurate assessment of radiation doses received by individuals during a radiological event such as a nuclear accident is crucial to containment and effective treatment. In the event of a large-scale radiological exposure, efficient prioritization of resources would require a fast and minimally-invasive biodosimetry assay. In this project we will assess the feasibility of two related methods for precise retrospective biodosimetry, and optimize experimental procedures to develop rapid, quantitative, and field-ready assays. The assays are based on our key observation that ionizing radiation generates specific, stable and quantifiable products from certain amino acid residues in proteins in a yield that is directly related to absorbed dose. Both traditional LCMS/MS and novel immunological detection methods will be developed for detection of the damaged residues from human blood proteins including hemoglobin and serum albumin. The LC-MS/MS quantification is eminently feasible and supported by preliminary data. The radiation chemical yield of the target analytes from authentic parent amino acids has been established by NMR and LC-MS/MS, with analytes detectable at doses as low as 10 Gy from milligram amounts of parent. This technique will provide precise measurements of product yield, and will allow analysis of factors that affect the yield and recovery of the analytes from biological samples. The prospect of using a proteomics approach in which LC MS/MS analysis of proteolyzed fragments will also be investigated. A complementary second method involves detection of analytes by immunological techniques, which could provide faster throughput, involve less sample preparation, and may ultimately be incorporated into an ELISA assay that would not require sophisticated instrumentation. Monoclonal antibody reagents specific for the analytes in peptide targets will be developed, using an antibody-phage display technology available at the University of Rochester. Both techniques benefit from the fact that the analyte is produced immediately following irradiation, eliminating the need for a waiting period before subjects can be reliably tested after exposure. Furthermore the products are stable and potentially can be measured in individuals for weeks or even months following exposure.

    Relevance: This research aims to develop a simple, accurate and rapid test for the amount of radiation damage sustained by an individual, such as may result from a nuclear accident or other radiation event. Such a test would allow rapid containment and treatment of affected individuals and is not currently available. We have characterized key products induced by radiation in proteins, and will use these to develop methodologies that will form the basis for a retrospective, simple, and rapid blood test that can be used in the field.

  14. ZTE imaging of teeth for EPR dosimetry applications[back to top]
    Gang Zhu, Ph.D. [PI]
    Department of MRI Application
    Bruker Biospin
    Billerica, MA
    Venkata Krishnamurthy Nemani, Ph.D [Co-PI]
    Department of Radiology
    The Geisel School of Medicine at Dartmouth

    Designated Liaison at Dart-Dose CMCR: Ben Williams

    Abstract: Electron paramagnetic resonance (EPR) is capable of estimating irradiation dose absorbed by teeth. Therefore, an EPR tooth biodosimeter should help effectively manage medical care for a large population that has been exposed to damaging radiation. Currently, the contribution of enamel thickness to EPR measurements is unknown, as are the contributions of dental fillings and caries.

    To quantitatively attribute EPR signal to irradiation dose alone, we propose to noninvasively and nondestructively image teeth using magnetic resonance imaging (MRI). Enamel thickness and tooth morphology, including dental fillings and caries, will be noted. These measurements will help accurately attribute EPR signal arising from absorbed radiation dose alone by removing the confounders of enamel thickness and other morphological parameters. Eventually, such measurements may help establish surrogate markers of enamel thickness that may be rapidly extracted in the field.

    Relevance: The proposed technique of using MRI to help quantitatively assign EPR signal to absorbed radiation dose in teeth should help assist in determining appropriate medical management of a very large population of people exposed to levels of radiation sufficient to cause acute radiation syndrome.

  15. Low Temperature ESR study of radiation-induced signals in nail clippings[back to top]
    Chandrasekhar Ramanathan, Sc.D. [PI]
    Department of Physics and Astrnomy
    Dartmouth College

    Designated Liaison at Dart-Dose CMCR: Steven G. Swarts, Ph.D

    Abstract: Electron spin resonance (or ESR) measurements of radiation-induced signals (RIS) in nail, teeth and bone have been proposed as a viable physically-based dosimetric technique for retrospective measurements of radiation exposure in the event of an accidental release of radiation or a terrorist attack. Ex vivo ESR measurements of fingernail clippings for radiation dosimetry are complicated by the presence of so-called mechanically-induced signals (MIS) that are produced during clipping of the fingernails. While the MIS signal can be eliminated by washing the nails with distilled water, this procedure also reduces the RIS signal. It has, however, been observed that a fraction of the radiation induced signal persists after the water treatment, and remains stable even under further treatment with water. Preliminary ESR experiments at Q-band (34 GHz) have revealed additional structure in the RIS singlet, suggesting that it might be possible to directly identify the long-lived species.

    The goal in this pilot project proposal is to measure and characterize the dose response of this long-lived residual signal in a W-band ESR experiment (94 GHz). By moving to high field and low temperature we will enhance both the sensitivity of the ESR experiment and the spectral resolution of the experiment. The use of pulsed ESR experiments will enable us to further elucidate information about the nature and stability of these radicals. Another second goal of this proposal is to compare this long-lived residual RIS signal to the signal present before the water treatment. In particular the ultimate goal remains to perform in vivo measurements of the RIS signal in the field. In vivo ESR measurements of the nail will not contain MIS signals and so the entire RIS signal will be observed. We will therefore characterize how the RIS signal changes following water treatment in order to better correlate the ex vivo studies with in vivo measurements.

    Relevance: The ability to rapidly screen large populations for acute radiation exposure is critical for guiding the response to industrial accidents or terrorist attacks with radiological weapons. Long-lived radiation-induced free-radicals in human teeth and nails can be detected by electron spin resonance, and these techniques could potentially be used to quantify the radiation dose that person has been exposed to and thus guide treatment.

  16. New step in sample preparation procedurefor ex-vivo EPR fingernail dosimetery[back to top]
    Stephen W.S. McKeever, Ph.D. [PI]
    Oklahoma State University
    Sergii Sholom, Ph.D. [Co-Investigator]
    Oklahoma Center for Radiation Physics
    Oklahoma State University
    Alexander Romanyukha, Ph.D. [Co-Investigator]
    Naval Dosimetry Center, US Navy, Bethesda, MD

    Designated Liaison at Dart-Dose CMCR: Steven G. Swarts, Ph.D

    Abstract: In the proposed project we will study human nails as a possible individual emergency electron paramagnetic resonance (EPR) dosimeter. The ultimate goal of this pilot project is to establish a procedure of sample preparation which will assure the dose measurements with accuracy and precision that are required for triage applications. The following experiments will be carried out:

    • a water-soaking treatment of fingernails prior to cutting will be tested in order to reduce the mechanically-induced EPR signals generated in nails during the cutting and increase accuracy of dose reconstruction technique with nails;
    • the nails will be studied in both X- and Q- microwave frequency bands. A Q-band, which has higher resolution than the X-band and is able to distinguish stable and unstable spectral components of EPR signals from nails, will be used to optimize the sample preparation procedures developed for the X-band, which is assumed to be used in triage applications;
    • an effect of nails pre-soaking on the stable and unstable components of the radiation-induced EPR signal (RIS) will be tested in order to reduce inter-sample variation of mechanically-induced signals and RIS;
    • optimization of the dose reconstruction protocol will be achieved through multiple experiments conducted on differently-treated fingernail samples collected from the same 10-15 individuals.

    From the outcomes of the above studies, we anticipate that a new version of emergency individual EPR dosimetry technique with nails will be developed and adopted for triage applications.

    Relevance: The main outcome of the project will be development of an emergency dosimetry technique with possibility of fast dose reconstruction for large number of people exposed due to a radiological accident, including a nuclear terrorism event, to provide them the necessary medical assistance, depending on the level of received radiation doses (triage application).

  17. A Clinical Analysis of Emesis as a Screening Diagnostic for Radiation Dose[back to top]
    Arif N.Ali, M.D, M.S [PI]
    Jeffrey Switchenko, Ph.D
    Emory University

    Designated Liaisons at Dart-Dose CMCR: Hal Swartz, M.D., Ph.D and Ann Barry Flood,Ph.D

    Abstract: In the event of a nuclear or radiologic disaster, it is of critical importance to quickly and accurately determine the radiation dose that patients have received for the purposes of triage and efficient allocation of limited medical resources. Current International Atomic Energy Agency (IAEA) guidelines, as well as various military manuals and emergency response textbooks, list the time-to-emesis as a key indicator of radiation dose. These recommendations are based, in large part, on radiation accident databases with inherently uncertain conditions and in the most widely used analysis, does not consider subjects receiving irradiation and who did not vomit. Although time-to-emesis is being widely recommended and implemented as a triage diagnostic for radiation dose, there has never been any formal medical literature analysis of emesis as a screening diagnostic (though a recent publication did report on a statistical analysis of time-to-emesis based on radiation accident data). Additionally, there has been no differentiation in time-to-emesis parameters between the high dose rate (initial detonation) and low dose rate exposures (fallout).

    We are proposing to undertake a systematic re-evaluation of the availability of data on time-to-emesis, adding in the experiences from therapeutic exposures. This initial proposal seeks to re-analyze a relatively recent, published clinical TBI and emesis data set (Westbrook et al., Clinical Radiology, 1987) to determine the sensitivity and specificity of time-to-emesis as a screening diagnostic for radiation dose from a low dose rate exposure scenario.

    Relevance: There have been recent publications analyzing time-to-emesis in radiation accident databases that have suggested that this indicator has the potential to be fraught with uncertainty and confounding factors. An analysis of time-to-emesis in human patients undergoing clinical radiation treatment with well characterized radiation administration and thoroughly documented side effects may be able to provide a definitive answer on the utility of time-to-emesis as an indicator of radiation dose.

  18. Development of X-band EPR cavities for invivo tooth dosimeter[back to top]
    Ke Wu, Ph.D. [PI]
    Beijing Institute of Radiation Medicine, P.R China

    Designated Liaisons at Dart-Dose CMCR: Ben Williams

    Abstract: In vivo EPR measurement of radiation induced signal from tooth enamel offers an attractive method to perform mass, early, quickly and in situ triage by victim's doses for facilitate medical decisions and treatment and most effectively take use of limited medical resources when large number of individuals suffer from ionizing radiation in radiation accidents. Traditional EPR spectroscopy is only suitable for retrospective tooth dosimetry for its invasively sampling. Recently new EPR spectroscopy method is developing for in vivo tooth measurements. L-band EPR in vivo biodosimeter has approached much practical feasibility, but X-band EPR for in vivo tooth dosimeter have less been developed. As a pilot study, this project aims to develop X-band EPR cavity used for in vivo tooth measurement, which is the core component of EPR spectroscopy and will mainly determine the feasibility and sensitivity of in vivo measurement.

    The cavity working with different microwave modes will be designed, constructed, manufactured and afterward the performance features be tested. The cavity has an aperture open on its wall. The microwave power can leak out from the aperture and apply on the tooth fixed in. The aperture runs through the cavity making external modulated magnetic field act to the tooth from both sides of the aperture. The cavity is designed by theoretical calculation and simulation methods first, and then manufactured. Some important features like microwave power and field modulation effects will be tested using DPPH sample by changing experiment power and modulation magnetic strength. The sensitivity for in vivo tooth dosimetry will be assessed by measuring whole piece of tooth samples irradiated with various doses. Several kinds of cavities working in different microwave mode will be studied in this project thereby to obtain the best cavity by comparing their performance features. Previous study has shown that it is reasonable to develop X-band EPR cavities for in vivo dosimetry.

    Relevance: This work will make X-band ESR much applicable for in vivo tooth dosimetry. It is actively helpful for rescue of large number people in radiation accidents.

  19. Intensity Standard for L-Band EPR Tooth Dosimetry[back to top]
    Periannan Kuppusamy, Ph.D. [PI]

    Designated Liaison at Dart-Dose CMCR:

    Abstract:Exposure of humans to large doses of high-energy radiation, either by accident or act of terrorism, is a severe risk with potential health implications. There is a great need for a quantitative method with a large throughput to measure the dose of radiation, at least retrospectively, to triage radiation-exposed population for treatment based on the exposure levels. EPR dosimetry is a potentially viable tool for rapid and noninvasive assessment of radiation exposure levels in human subjects. The assessment requires the use of a reliable paramagnetic reference sample (standard) for quantitation of the observed EPR signal intensity from irradiated tooth. This Pilot Project will develop such an intensity reference standard which is robust, stable, and does not interfere with the EPR spectrum of tooth exposed to radiation. The availability of the reference material will enhance our ability to estimate the dose levels accurately using EPR dosimetry.

    The following specific aims are proposed:

    1. Prepare crystalline forms of paramagnetic Tempol in a diamagnetic host matrix.The material will be prepared as large crystals. The EPR spectra of the crystals will be characterized in single-crystal form as well as in polycrystalline form. An optimum level of dilution will be determined for best signal intensity, and resolution of lineshape.
    2. Characterize the EPR properties of single and polycrystalline (powder) forms of the material.We will characterize the EPR properties of single as well as polycrystalline (powder) forms of the material for anisotropy in the g- and hyperfine coupling constants and select the optimum material for use as reference standard for dosimetry.
    3. Encapsulate the single crystal, or polycrystalline powder in biocompatible polymers.We will perform thorough physicochemical and EPR characterization of the encapsulated material.

    Relevance:Electron paramagnetic resonance (EPR) tooth dosimetry is an attractive approach for dose estimation for initial triage as well as to guide subsequent treatment of people exposed to high-energy radiation. The goal of the Dart-Dose CMCR program is to develop a field-deployable physical biodosimetry EPR dosimeter. To be useful in an emergency situation, such as would occur in the aftermath of a radiation accident or exposure, the system should be robust and provide reliable data that can be used to calculate the dose accurately. The calculation requires the EPR intensity data collected from the irradiated tooth to be corrected to account for any variability in the instrument/measurement settings. This is done using a reference standard, a paramagnetic material with known EPR properties, placed at a reproducible position within the active volume of the resonator. EPR measurements will capture both the signal from the tooth and the reference material simultaneously. The accuracy of the dose estimation strongly depends on the stability and reproducibility of the reference standard. The goal of this pilot project is to make available a well-defined and stable reference standard that will improve the precision of the measurements and permit greater confidence in quantifying for triage purposes the dose received.