21st Century Biodosimetry: Quantifying the Past and Predicting the Future Symposium
February 22, 2001
Introduction
R. Julian Preston, Program Chair
Gene Expression Profiles for Monitoring Radiation Exposure
Sally A. Amundson, National Cancer Institute
We have previously demonstrated that stress gene responses to genotoxic agents vary widely in cell lines from different tissues of origin and different genetic backgrounds, highlighting the importance of cellular context to stress responses. In the specific case of ionizing radiation, details of exposure such as dose, dose rate, radiation quality, and elapsed time also result in variations in the observed response. The premise will be developed that stress gene response signatures may be employed as molecular markers for radiation exposure using a combination of informatics and functional genomics approaches. The key will be defining sets of genes that are informative for different outcomes of interest. A generalized post- exposure profile may aid in identifying exposed individuals within a population. More specific fingerprints may possibly be identified to reveal the specific agents, timing, and approximate dose of a radiation exposure. Previously, we have demonstrated changes in gene expression in human cell lines following doses of gamma rays as low as 2 cGy. Then, as a first step to in vivo studies in humans, we have measured gene induction in peripheral blood lymphocytes irradiated ex vivo with doses as low as 20 cGy. More recently, we have found diverse genes elevated in vivo 24 h after 20 cGy whole body irradiation of mice. These studies should provide insight into the molecular responses to physiologically relevant doses that cannot necessarily be extrapolated from high-dose studies. Ongoing microarray analyses meanwhile continue to identify large numbers of potential biomarkers from varied irradiation protocols. Computation-intensive informatics analysis methods are being developed for management of the complex gene expression profiles resulting from these experiments. As such work continues, it may be possible to identify individual variation in post-exposure profiles that correlate with the outcome of exposure. One example of this might be identifying patients likely to fail radiotherapy from their gene expression profile following the first fraction, thus allowing alternate therapies to be begun at an earlier stage of treatment. In the case of occupational or environmental exposures, such profiles may identify those individuals most susceptible to long term effects. A significant amount of data will be needed to develop meaningful correlations with various risks, however, and any suspected correlation between profile and outcome must be established and tested before this tool is of practical diagnostic use.
Quantification of Chromosome Abnormalities Using the Polymerase Chain Reaction
Gino A. Cortopassi, University of California
The Polymerase Chain Reaction (PCR) has the ability to detect rare chromosomal rearrangements in human blood or tissue samples with high sensitivity. One cause of nuclear chromosomal rearrangements is ionizing radiation, and specific rearrangements of nuclear oncogenes are common in hematological malignancies. Some evidence indicates that ionizing radiation of cells causes an increase in nuclear chromosomal rearrangements which are detectable by the PCR. Also, most cells contain from 10 to 2,000 copies of the mitochondrial genome, or mtDNA, and so the frequency of rearrangements of this repeated genome can also be investigated in response to radiation. There is substantial evidence for increased mtDNA mutagenesis as a result of exposure to nonionizing radiation, and some evidence for increased mutagenesis of mtDNA as a result of exposure to ionizing radiation. Baseline levels of mtDNA deletion are detectable in all tissues, and rise with age. The current status of PCR as a means to detect and quantify nuclear and mitochondrial chromosome rearrangements as a result of ionizing and non-ionizing radiation will be discussed.
Radiation Exposure Assessment Using Cytological and Molecular Biomarkers
William F. Blakely, Armed Forces Radiobiology Research Institute
New radiation exposure assessment methodologies being developed by the Armed Forces Radiobiology Research Institute (AFRRI) use the latest advances in molecular biotechnology to identify radiation-responsive cytogenetic and molecular biomarkers. The methodologies could eventually make it possible for field laboratories to assess radiation exposure doses from blood samples, using battery-operated, hand-held devices. Together with our diagnostic software also in development, the methodologies will improve the United States military’s emergency response capability and medical readiness. Radiation exposure assessment is conventionally determined based on peripheral blood lymphocyte depletion and on chromosome aberration detection. We have recently established an alternative methodology for measuring radiation-induced chromosome aberrations in interphase cells (Mut. Res. 466, 131-141, 2000). The method uses commercially available chemical agents to induce premature chromosome condensation in about 40 percent of resting G0 human peripheral blood lymphocytes. Then specific whole-chromosome DNA hybridization probes are used with fluorescence in situ hybridization to rapidly detect damaged cells over a broad dose range. In the area of peripheral blood lymphocyte depletion, we are evaluating two small-footprint commercial blood cell counters. In addition to those methods, we recently identified radiation-responsive gene expression targets and DNA mutations that can be analyzed using the rapid, real-time fluorogenic 5′-nuclease, or TaqManTM, polymerase chain reaction assay. Our goal is to establish rapid, high-throughput systems that provide precise assessments and are practical in a variety of radiation exposure scenarios. Our Biodosimetry Assessment Tool software application will equip health care providers with diagnostic information (clinical signs and symptoms, physical dosimetry, etc.) germane to the management of human radiation casualties. Designed primarily for prompt use after a radiation incident, the user-friendly program facilitates collection, integration, and archiving of data obtained from accidentally exposed persons.
Abortive Base Excision Repair of Radiation-Induced Clustered Lesions: A Concern for Risk Assessment
Susan S. Wallace, University of Vermont
Energy from low-LET ionizing radiation, such as x and gamma rays, is deposited in the water surrounding the DNA molecule such that between two to five radical pairs are generated within a radius of one to four nanometers. As a result, multiple single lesions, including oxidized purine or pyrimidine bases, sites of base loss, and single strand breaks, can be formed in DNA from the same radiation energy deposition event. The single lesions in these so-called multiply damaged sites or clustered lesions are repaired by base excision repair. Here we show that clustered DNA damages are formed in bacterial cells by ionizing radiation and are converted to lethal double strand breaks during attempted repair. In wild type cells possessing the oxidative DNA glycosylases that recognize and cleave DNA at repairable single damages, double strand breaks are formed at radiation-induced clusters during postirradiation incubation and in a dose-dependent fashion. Mutant cells lacking these enzymes do not form double strand breaks postirradiation and are substantially more radioresistant than wild type cells. These radioresistant mutant cells can be made radiosensitive by overexpressing one of the oxidative DNA glycosylases. Thus the effect of the oxidative DNA glycosylases in potentiating DNA damage must be considered when estimating radiation risk.
Clustered DNA Damages Induced by Low Doses of Ionizing Radiation
Betsy M. Sutherland, Brookhaven National Laboratory
Ionizing radiation induces clusters of DNA damages— oxidized bases, abasic sites, and strand breaks—on opposing strands within a few helical turns. Such damages have been postulated to be difficult to repair, as are double strand breaks (one type of cluster). We have shown that low doses of low and high-LET radiation induce such damage clusters, as well as isolated damages, in DNA in solution in human cells (Sutherland et al., Proc. Natl. Acad. Sci. 97, 103-108, 2000; Biochemistry 39, 8026-8031, 2000). Double strand breaks comprise only about 20 percent of such complex damages, with other cluster types being about 80 percent. The dose-response line for cluster induction is a straight line on a log-log plot with a slope of ~1, showing that a cluster is produced by one radiation “hit,” a particle or photon plus its accompanying track. This implies that even low doses of ionizing radiation can produce clustered damages. Studies are in progress to determine whether clusters can be produced by mechanisms other than ionizing radiation.
Discussion
Biomarkers of Exposure and Dose: State of the Art
Antone L. Brooks, Washington State University
Current data on biodosimetry and potential future research directions will be outlined to provide advice to the Defense Threat Reduction Agency. Biomarkers of exposure and dose have two major uses. First, they must rapidly tell the field commanders the personnel exposure level and determine which individuals may require medical attention. This application does not require high sensitivity but rapid turn-around is essential for proper decision making. Many molecular techniques such as changes in gene expression, meet these requirements and can be used under combat conditions. Second, biodosimetry can be used to evaluate the dose and risks for late effects. Here, turn around time is not critical but sensitivity must be high. Chromosome aberrations meet the sensitivity requirements for evaluation of low doses and their associated risks. Such measures help the troops understand the magnitude of potential cancer risk and provide major positive psychological impact. Newer methods of detection and automation of biomarkers will be discussed.
Validated Biomarker Responses May Influence Medical Surveillance of Individuals Exposed to Genotoxic Agents
Richard J. Albertini, University of Vermont
There is currently a vast armamentarium of biomarkers for evaluating human exposures to genotoxic agents, the effects of such exposures and/or susceptibility to any deleterious consequences. Before application, however, these biomarkers require validation in terms of truly reflecting what is claimed. Transitional epidemiological studies bridge the gap between laboratory and field. In a transitional study, a biomarker response is the dependent variable being evaluated, while the intended measure, i.e., exposure, effect or susceptibility, is the independent variable. Once validated, biomarker responses provide valuable data for use in making human health risk assessments and as guides for individual medical surveillance programs. An analysis of medical decision making illustrates how biomarker responses that increase the relative risk of subsequent disease occurrence change “pre-test likelihoods,” thereby influencing interpretation of medical diagnostic tests and even the choice of tests to be performed. This argues that an individual’s response using validated biomarkers should be made part of the medical record.
FISH Cytogenetics and the Future of Radiation Biodosimetry
James D. Tucker, Lawrence Livermore National Laboratory
Fluorescence in situ hybridization (FISH) with whole chromosome paints has greatly facilitated the analysis of structural aberrations and has led to translocations replacing dicentrics as the aberration of choice. Major challenges remain if we are to go from translocations to an understanding of the health consequences of exposure. Yet to be surmounted are the roles of individual susceptibility, time since exposure, age, and the role of adaptation to radiation. Accomplishing these objectives will require automation, reduced costs, improved calibration and extensive use of baseline samples.
Evaluation of Three Somatic Genetic Biomarkers as Indicators of Low-Dose Radiation Effects in Cleanup Workers of the Chernobyl Nuclear Reactor Accident
Irene M. Jones, Lawrence Livermore National Laboratory
The exposure of >600,000 people during the clean up effort after the 1986 Chernobyl accident has posed a challenge and an opportunity for the radiobiology community. This report will present results and implications of a large study in which GPA mutants did not detect a radiation exposure effect, while chromosome translocations and HPRT mutations detected an effect, translocations being more sensitive.
Is there the Potential for Biomarkers that are Specific to High-LET Radiation?
David J. Brenner, Columbia University
There have been several suggestions of biomarkers that are specific to high-LET radiation. Such a biomarker could significantly increase the power of epidemiological studies of individuals exposed to densely-ionizing radiations such as alpha particles (e.g., radon, plutonium workers) or neutrons (e.g., DOE/NRC workers, airline personnel). We discuss here a potential high-LET biomarker which is the ratio of induced intra-arm to inter-chromosomal aberrations. Both theoretical and experimental studies have suggested that this ratio will be different by a factor of about three between high-LET and any other likely clastogen. Because both types of aberrations (intra-arm and inter-chromosomal aberrations) are (1) frequent and (2) measurable at long times after exposure, this ratio appears to be a practical biomarker of high-LET exposure. Evidence of the long-term stability of this chromosomal biomarker has also been generated. Several groups (Munich, Livermore, and Columbia) have all validated this LET fingerprint in vitro for unstable aberrations, but the technology for efficiently measuring the corresponding stable intra-arm aberrations (paracentric inversions) is only now becoming available.
Program Committee:
R. Julian Preston, Chair
Richard J. Albertini
David J. Brenner
Antone L. Brooks
James D. Tucker
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