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Although it is documented that concurrent wounding increases mortality from radiation injury, the molecular mechanism of combined injury is unknown. In this study, mice were exposed to gamma radiation followed by skin wounding. Wound trauma exacerbated radiation-induced mortality, reducing the LD(50/30) from 9.65 Gy to 8.95 Gy. Analyses of histopathology, inducible nitric oxide synthase (iNOS), and serum cytokines were performed on mouse ileum and skin at various times after 9.75 Gy and/or wounding. In the ileum, the villi were significantly shortened 3 days postirradiation but not after wounding; combined injury resulted in decreased villus width and tunica muscularis thickness. The skin of mice subjected to combined injury was less cellular and had a smaller healing bud than the skin of mice subjected to wounding alone. Combined injury significantly delayed wound closure times; it also prolonged the increased levels of iNOS protein in the skin and ileum. iNOS up-regulation was correlated with increases in transcription factors, including NF-kappaB and NF-IL6. The increase in NF-IL6 may be due to increases in cytokines, including IL-1beta, -6, -8, -9, -10 and -13, G-CSF, eotaxin, INF-gamma, MCP-1, MIP-1alpha and MIP-1beta. Combined injury resulted in early detection of bacteria in the blood of the heart and liver, whereas radiation alone resulted in later detection of bacteria; only a transient bacteremia occurred after wounding alone. Results suggest that enhancement of iNOS, cytokines and bacterial infection triggered by combined injury may contribute to mortality. Agents that inhibit these responses may prove to be therapeutic for combined injury and may reduce related mortality.

Partial-body biodosimetry is likely to be required after a radiological or nuclear exposure. Clinical signs and symptoms, distribution of dicentrics in circulating blood cells, organ-specific biomarkers, and physical signals in teeth and fingernails all can provide indications of non-homogeneous exposures. Organ specific biomarkers may provide early warning regarding physiological systems at risk after radiation injury. Use of a combination of markers and symptoms will be needed for clinical insights for therapeutic approaches. Analysis of dicentrics, a marker specific for radiation injury, is the "gold standard" of biodosimetry and can reveal partial-body exposures. Automation of sample processing for dicentric analysis can increase throughput with customization of off-the-shelf technologies for cytogenetic sample processing and information management. Automated analysis of the metaphase spreads is currently limited, but improvements are in development. The efforts described here bridge the technological gaps to allow the use of dicentric chromosome assay (DCA) for risk-based stratification of mass casualties. This article summarizes current knowledge on partial-body cytogenetic dose assessment, synthesizing information leading to the proposal of an approach to triage dose prediction in radiation mass casualties that is based on equivalent whole-body doses under partial-body exposure conditions and assesses the validity of using this model. An initial screening using only 20 metaphase spreads per subject can confirm irradiation above 2 Gy. A subsequent increase to 50 metaphases improves dose determination to allow risk stratification for clinical triage. Metaphases evaluated for inhomogeneous distribution of dicentrics can reveal partial-body exposures. The authors tested the validity of this approach in an in vitro model that simulates partial-body irradiation by mixing irradiated and un-irradiated lymphocytes in various proportions. Preliminary results support the notion that this approach will be effective under a range of conditions including some partial-body exposures, but may have limitations with low doses or small proportions of irradiated parts of the body. These studies address an important problem in the diagnosis of partial-body irradiation and dose assessment in mass casualties and propose a solution. However, additional work is needed to fully develop and validate the application of DCA to partial-body exposures.

Straume, T., Amundson, S. A., Blakely, W. F., Burns, F. J., Chen, A., Dainiak, N., Franklin, S., Leary, J. A., Loftus, D. J., Morgan, W. F., Pellmar, T. C., Stolc, V., Turteltaub, K. W., Vaughan, A. T., Vijayakumar, S. and Wyrobek, A. J. NASA Radiation Biomarker Workshop. September 27–28, 2007. Radiat. Res. 170, 393–405 (2008). A summary is provided of presentations and discussions at the NASA Radiation Biomarker Workshop held September 27–28, 2007 at NASA Ames Research Center in Mountain View, CA. Invited speakers were distinguished scientists representing key sectors of the radiation research community. Speakers addressed recent developments in the biomarker and biotechnology fields that may provide new opportunities for health-related assessment of radiation-exposed individuals, including those exposed during long-duration space travel. Topics discussed included the space radiation environment, biomarkers of radiation sensitivity and individual susceptibility, molecular signatures of low-dose ...

Excessive generation of free radicals has been implicated in several pathological conditions. We demonstrated previously that peroxide-generated free radicals decrease calcium-dependent high K+-evoked L-[exp 3H]-glutamate release from synaptosomes while increasing calcium-independent basal release. The present study evaluates the nonvesicular release of excitatory amino acid neurotransmitters, using D-[exp 3H]aspartate as an exogenous label of the cytoplasmic pool of L-glutamate and L-aspartate. Isolated presynaptic nerve terminals from the guinea pig cerebral cortex were used to examine the actions and interactions of peroxide, iron, and desferrioxamine. Pretreatment with peroxide, iron alone, or peroxide with iron significantly increased the calcium-independent basal release of D-[exp 3H]aspartate. Pretreatment with desferrioxamine had little effect on its own but significantly limited the enhancement by peroxide. High K+-evoked release in the presence of Ca(exp 2+) was enhanced by peroxide but not by iron. These data suggest that peroxide increases nonvesicular basal release of excitatory amino acids through Fenton-generated hydroxyl radicals. This release could cause accumulation of extracellular excitatory amino acids and contribute to the excitotoxicity associated with some pathologies.

Ionizing radiation causes striking changes in hippocampal activity in vivo. Changes in neuronal firing patterns and spiking activity in electroencephalographic recordings appear at doses as low as 4 Gy. Accompanying exposure to ionizing radiation is a breakdown in blood brain barrier and a decrease in cerebral blood flow. In an effort to define the mechanisms of radiation damage to neuronal excitability, without these complicating factors, the effects of radiation on neuronal activity in hippocampal slices were investigated. Damage is likely to result from generation of free radicals. Since peroxide mixed with iron produces hydroxyl free radicals through the Fenton reaction, peroxidative damage was evaluated on hippocampal slices for comparison.

Current events throughout the world underscore the growing threat of different forms of terrorism, including radiological or nuclear attack. Pharmaceutical products and other approaches are needed to protect the civilian population from radiation and to treat those with radiation-induced injuries. In the event of an attack, radiation exposures will be heterogeneous in terms of both dose and quality, depending on the type of device used and each victim's location relative to the radiation source. Therefore, methods are needed to protect against and treat a wide range of early and slowly developing radiation-induced injuries. Equally important is the development of rapid and accurate biodosimetry methods for estimating radiation doses to individuals and guiding clinical treatment decisions. Acute effects of high-dose radiation include hematopoietic cell loss, immune suppression, mucosal damage (gastrointestinal and oral), and potential injury to other sites such as the lung, kidney and central nervous system (CNS). Long-term effects, as a result of both high- and low-dose radiation, include dysfunction or fibrosis in a wide range of organs and tissues and cancer. The availability of appropriate types of animal models, as well as adequate numbers of animals, is likely to be a major bottleneck in the development of new or improved radioprotectors, mitigators and therapeutic agents to prevent or treat radiation injuries and of biodosimetry methods to measure radiation doses to individuals.

To help the nation prepare for the possibility of a terrorist attack using radiological and nuclear devices, the Office of Science and Technology Policy and the Homeland Security Council established an interagency working group. The working group deliberated on the research needs for radiological/ nuclear threat countermeasures and identified and prioritized 18 areas for further attention. The highest priorities were given to research on (1) radioprotectors for use prior to exposure; (2) therapeutic agents for postexposure treatment; (3) antimicrobial therapy for infections associated with radiation exposure; (4) cytokines and growth factors; (5) mechanisms of radiation injury at the molecular, cellular, tissue and organism levels; and (6) automation of biodosimetric assays. High priority was given to (1) developing biomarkers for biodosimetry; (2) enhancing training in the radiation sciences; (3) exploring the consequences of combined injury; (4) establishing a repository of information regarding investigational countermeasures; and (5) following the health of an exposed population to better prepare for subsequent events. The research areas that the committee felt required the attention of the radiation research community are described in this report in an effort to inform this community about the needs of the nation and to encourage researchers to address these critical issues.

To develop approaches to prophylaxis/protection, mitigation and treatment of radiation injuries, appropriate models are needed that integrate the complex events that occur in the radiation-exposed organism. While the spectrum of agents in clinical use or preclinical development is limited, new research findings promise improvements in survival after whole-body irradiation and reductions in the risk of adverse effects of radiotherapy. Approaches include agents that act on the initial radiochemical events, agents that prevent or reduce progression of radiation damage, and agents that facilitate recovery from radiation injuries. While the mechanisms of action for most of the agents with known efficacy are yet to be fully determined, many seem to be operating at the tissue, organ or whole animal level as well as the cellular level. Thus research on prophylaxis/protection, mitigation and treatment of radiation injuries will require studies in whole animal models. Discovery, development and delivery of effective radiation modulators will also require collaboration among researchers in diverse fields such as radiation biology, inflammation, physiology, toxicology, immunology, tissue injury, drug development and radiation oncology. Additional investment in training more scientists in radiation biology and in the research portfolio addressing radiological and nuclear terrorism would benefit the general population in case of a radiological terrorism event or a large-scale accidental event as well as benefit patients treated with radiation.

An Institute of Medicine committee was convened to explore the links between biological, psychosocial, and behavioral factors and health and to review effective applications of behavioral interventions. Based on the evidence about interactions of the physiological responses to stress, behavioral choices, and social influences, the committee encouraged additional research efforts to explore the integration of these variables and to evaluate their mechanisms. An understanding of the social factors influencing behavior is growing and should be considered in programs and policies for public health, in addition to individual behavior and physiological status. Interventions to change behaviors have been directed toward individuals, communities, and society. Many intervention trials have documented the capacity of interventions to modify risk factors. However, more trials that include measures of morbidity and mortality to determine if the strategy has the desired health effects are needed. Behavior can be changed and new behaviors can be taught. Maintaining behavior changes is a greater challenge. Although short-term changes in behavior following interventions are encouraging, long-duration efforts are needed to improve health outcomes and to provide long-term assessments of effectiveness. Interventions aimed at any level can influence behavior change; however, existing research suggests that concurrent interventions at multiple levels are most likely to sustain behavior change and should be encouraged.

The Persian Gulf War resulted in injuries of US Coalition personnel by fragments of depleted uranium (DU). Fragments not immediately threatening the health of the individuals were allowed to remain in place, based on long-standing treatment protocols designed for other kinds of metal shrapnel injuries. However, questions were soon raised as to whether this approach is appropriate for a metal with the unique radiological and toxicological properties of DU. The Armed Forces Radiobiology Research Institute (AFRRI) is investigating health effects of embedded fragments of DU to determine whether current surgical fragment removal policies remain appropriate for this metal. These studies employ rodents implanted with DU pellets as well as cultured human cells exposed to DU compounds. Results indicate uranium from implanted DU fragments distributed to tissues far-removed from implantation sites, including bone, kidney, muscle, and liver. Despite levels of uranium in the kidney that were nephrotoxic after acute exposure, no histological or functional kidney toxicity was observed. However, results suggest the need for further studies of long-term health impact, since DU was found to be mutagenic, and it transformed human osteoblast cells to a tumorigenic phenotype. It also altered neurophysiological parameters in rat hippocampus, crossed the placental barrier, and entered fetal tissue. This report summarizes AFRRI's depleted uranium research to date.

During the 1991 Persian Gulf War several US military personnel were wounded by shrapnel fragments consisting of depleted uranium. These fragments were treated as conventional shrapnel and were not surgically removed to spare excessive tissue damage. Uranium bioassays conducted over a year after the initial uranium injury indicated a significant increase in urine uranium levels above natural background levels. The potential mutagenic effects of depleted uranium are unknown. To assess the potential mutagenic effects of long-term exposure to internalized depleted uranium, Sprague-Dawley rats were implanted with depleted uranium and their urine and serum were evaluated for mutagenic potential at various times after pellet implantation using the Ames Salmonella reversion assay. Tantalum, an inert metal widely used in prosthetic devices was used for comparison. Enhancement of mutagenic activity in Salmonella typhimurium strain TA98 and the Ames II mixed strains (TA7001-7006) was observed in urine samples from animals implanted with depleted uranium pellets. In contrast, urine samples from animals implanted with tantalum did not show a significant enhancement of mutagenic activity in these strains. In depleted uranium-implanted animals, urine mutagenicity increased in a dose- and time-dependent manner demonstrating a strong positive correlation with urine uranium levels (r = 0.995, P0.001). There was no mutagenic enhancement of any bacterial strain detected in the sera of animals implanted with either depleted uranium or tantalum pellets. The results suggest that uranium content in the urine is correlated with urine mutagenicity and that urinary mutagenicity might be used as a biomarker to detect exposure to internalized uranium.

Metabolic integrity of glial cells in field CA1 of the guinea pig hippocampus is critical to maintenance of synaptic transmission (Keyser and Pellmar [1994] Glia 10:237-243). To determine if this tight glial-neuronal coupling is equally important in other brain regions, we compared the effect of fluoroacetate (FAC), a glial specific metabolic blocker, on synaptic transmission in field CA1 to synaptic transmission in area dentata (DG). FAC was significantly more effective in decreasing synaptic potentials in CA1 than in DG. A similar regional disparity in the FAC-induced decrease in ATP levels was evident. Isocitrate, a glial specific metabolic substrate, prevented the FAC-induced synaptic depression in both CA1 and DG. The results suggest that glia of CA1 and dentate respond differently to metabolic challenge. Modulation of this glial-neuronal coupling could provide a regionally specific mechanism for synaptic plasticity. Additionally, site-specific glial-neuronal interactions can impact on a variety of physiological and pathophysiological conditions.

RIPD (Radiation-Induced Performance Decrement) is a physiologically-based model of acute radiation sickness (ARS). RIPD estimates: severity of illness due to prompt or protracted exposures to ionizing radiation for total free-in-air doses between 0.75 Gy and 45 Gy; residual performance capability over time for soldiers exposed to ionizing radiation in a military engagement; incidence of performance decrement; incidence of lethality; time to lethality. The RIPD model applies to whole-body exposures to gamma rays and/or neutrons; dose-rate histories can be complex and the exposure period can be as long as one week. RIPD estimates severity of illness and performance capability for up to 1000 hours (about 6 weeks) after the start of exposure. The prompt exposure models in RIPD were developed during the Defense Nuclear Agency's Intermediate Dose Program (IDP) to facilitate consequence assessment and military planning in a nuclear radiation environment. This report (1) summarizes RIPD's performance decrement model, and (2) reviews the four kinetic models around which RIPD's sign/symptom (S/S) severity model structure is built and through which RIPD extends the IDP's prompt exposure S/S severity models to protracted radiation exposures. The assumptions, limitations, and mathematical structure of these four physiologically-based models are discussed.

The health effects of embedded fragments of depleted uranium (DU) are being investigated to determine whether current surgical fragment-removal policies are appropriate for this metal. The authors studied rodents implanted with DU pellets as well as cultured human cells exposed to DU compounds. Results indicate that uranium from implanted DU fragments distributes to tissues distant from implantation sites, including bone, kidney, muscle, and liver. Despite levels of uranium in kidney that would be nephrotoxic after acute exposure, no histological or functional kidney toxicity was observed with embedded DU, indicating that the kidney adapts when exposed chronically. Nonetheless, further studies of the long-term health impact are needed. DU is mutagenic and transforms human osteoblastic cells into a tumorigenic phenotype. It alters neurophysiological parameters in rat hippocampus, crosses the placental barrier, and enters fetal tissue. Preliminary data also indicate decreased rodent litter size when animals are bred 6 months or longer after DU implantation.

Biological dosimetry is an essential tool for estimating radiation dose. The dicentric chromosome assay (DCA) is currently the tool of choice. Because the assay is labor-intensive and time-consuming, strategies are needed to increase throughput for use in radiation mass casualty incidents. One such strategy is to truncate metaphase spread analysis for triage dose estimates by scoring 50 or fewer metaphases, compared to a routine analysis of 500 to 1000 metaphases, and to increase throughput using a large group of scorers in a biodosimetry network. Previously, the National Institutes for Allergies and Infectious Diseases (NIAID) and the Armed Forces Radiobiology Research Institute (AFRRI) sponsored a double-blinded interlaboratory comparison among five established international cytogenetic biodosimetry laboratories to determine the variability in calibration curves and in dose measurements in unknown, irradiated samples. In the present study, we further analyzed the published data from this previous study to investigate how the number of metaphase spreads influences dose prediction accuracy and how this information could be of value in the triage and management of people at risk for the acute radiation syndrome (ARS). Although, as expected, accuracy decreased with lower numbers of metaphase spreads analyzed, predicted doses by the laboratories were in good agreement and were judged to be adequate to guide diagnosis and treatment of ARS. These results demonstrate that for rapid triage, a network of cytogenetic biodosimetry laboratories can accurately assess doses even with a lower number of scored metaphases.

Previous studies in our laboratory have suggested that an oxidation reaction is responsible for the actions of free radicals to decrease synaptic potentials. Recently we observed that free radicals both decreased depolarization-induced vesicular release and enhanced basal, nonvesicular release of the excitatory amino acid, [3H]L-glutamate. In order to evaluate the contribution of oxidative reactions to this latter effect, we evaluated the actions of the oxidizing agent chloramine-T on synaptosomal release of excitatory amino acids, using [3H]D-aspartate as the exogenous label. Basal and depolarization evoked [3H]D-aspartate release were calcium-independent and nonvesicular. Chloramine-T pretreatment significantly increased basal release, while having no effect on high K+-evoked release. These data suggest that an oxidative process can mimic the free radical increase of basal release, as well as the decrease in synaptic potentials. On the other hand, the calcium-independent-evoked release may involve a different mechanism. Our results demonstrate that under basal, nondepolarizing conditions, oxidative stress exerts an adverse effect on the presynaptic nerve terminal, resulting in an increased release of potentially damaging excitatory amino acid neurotransmitters.

Radiation exposures from accidents, nuclear detonations or terrorist incidents are unlikely to be homogeneous; however, current biodosimetric approaches are developed and validated primarily in whole-body irradiation models. A workshop was held at the Armed Forces Radiobiology Research Institute in May 2008 to draw attention to the need for partial-body biodosimetry, to discuss current knowledge, and to identify the gaps to be filled. A panel of international experts and the workshop attendees discussed the requirements and concepts for a path forward. This report addresses eight key areas identified by the Workshop Program Committee for future focus: (1) improved cytogenetics, (2) clinical signs and symptoms, (3) cutaneous bioindicators, (4) organ-specific biomarkers, (5) biophysical markers of dose, (6) integrated diagnostic approaches, (7) confounding factors, and (8) requirements for post-event medical follow-up. For each area, the status, advantages and limitations of existing approaches and suggestions for new directions are presented.

Basal (non-depolarized) and high K + -stimulated [ 3 H] l -glutamate release in the presence and absence of Ca 2+ were assessed using presynaptic nerve terminals (synaptosomes) isolated from the cerebral cortex of the guinea pig. Basal glutamate release was found to be Ca 2+ -independent and was significantly increased following treatment with hydrogen peroxide (H 2 O 2 ). On the other hand, depolarization-induced release had both a Ca 2+ -dependent and Ca 2+ -independent component. Both components of stimulated release were suppressed by H 2 O 2 . In fact, Ca 2+ -dependent evoked release was virtually eliminated by H 2 O 2 pretreatment. The data suggest that H 2 O 2 exerts a differential effect on the neurochemical mechanisms involved in basal and stimulated glutamate release at the presynaptic nerve terminal.

In an effort to understand the damaging actions of free radicals to neuronal electrophysiology, the superoxide generator, dihydroxyfumarate (DHF), was evaluated in slices of guinea pig hippocampus. Using field potential recording techniques, population spikes and population synaptic potentials were recorded in field CA1. Slices were exposed to 3 mM DHF either alone or in the presence of a protectant. DHF did not alter the ability of the afferent volley to generate a synaptic potential, but it did impair the ability of the synaptic potential to elicit a population spike. In addition, DHF induced lipid peroxidation as measured by the thiobarbituric acid assay. Superoxide dismutase (SOD) provided no protection. Instead, SOD treatment promoted DHF damage to synaptic potentials. Catalase alone mitigated the actions of DHF, but only in SOD plus catalase was the DHF-induced electrophysiological deficit and lipid peroxidation completely antagonized. The iron chelator, Desferal, did not protect but promoted synaptic damage. Desferal may be ineffective because of the nitroxide radical formed upon its reaction with DHF. The hydroxyl radical scavenger, dimethylsulfoxide, prevented lipid peroxidation and reduced the DHF-induced deficit but did not completely prevent the impairment of spike generation. These data suggest that DHF exerts its actions through generation of hydrogen peroxide which would further react with tissue iron to produce hydroxyl radicals.

Casualties of radiation dispersal devices, nuclear detonation or major ionizing radiation accidents, in addition to radiation exposure, may sustain physical and/or thermal trauma. Radiation exposure plus additional tissue trauma is known as combined injury. There are no definitive therapeutic agents. Cyclooxygenase-2 (COX-2), an inducible enzyme expressed in pathological disorders and radiation injury, plays an important role in inflammation and the production of cytokines and prostaglandin E(2) (PGE(2)) and could therefore affect the outcome for victims of combined injury. The COX-2 inhibitors celecoxib and meloxicam were evaluated for their therapeutic value against combined injury in mice. In survival studies, the COX-2 inhibitors had no beneficial effect on 30-day survival, wound healing or body weight gain after radiation injury alone or after combined injury. Meloxicam accelerated death in both wounded and combined injury mice. These drugs also induced severe hepatic toxicity, exaggerated inflammatory processes, and did not enhance hematopoietic cell regeneration. This study points to potential contraindications for use of COX-2 inhibitors in patients undergoing therapy for radiation injury and combined injury.

Medical countermeasures are required to address the emerging radiological and nuclear threats. Radiation injury can result from exposure to external, penetrating radiation or internalized radionuclides. Countermeasures for internal contamination focus on decorporation and protecting the primary site of activity. No countermeasures are currently approved for the acute radiation syndrome that results from external exposures, but significant efforts are ongoing to develop new agents to counter the cascade of damage from ionizing radiation. Antioxidants and enzyme mimics are directed toward prevention of free radical damage to macromolecules. Other agents target the affected cell-signaling pathways: preventing apoptosis, triggering cell cycle arrest, or modulating the inflammatory response. To counter the loss of progenitor cells, particularly in the bone marrow and the gut, drugs such as the cytokines and other immunomodulators are developed to promote proliferation of new cells. If radiation injury proceeds to organ failure and infection, supportive care and antibiotics provide significant therapeutic value. In addition to the radiation dose, several factors impact the severity of the injury. Among these are radiation quality (e.g. neutron–gamma mixed fields) and dose rate, combined injuries, and partial-body exposures. In future efforts, it will be important to consider these factors, which can modulate the radiation response and possibly affect the efficacy of the treatments. Although an ideal countermeasure that is inexpensive, stable with a long shelf-life, easily administered, nontoxic, and efficacious is yet to be attained, new options continue to arise. Many promising drugs are in the pipeline. Keywords: radiation countermeasures; acute radiation syndrome; internal contamination; decorporation

Non-clinical human radiation exposure events such as the Hiroshima and Nagasaki bombings or the Chernobyl accident are often coupled with other forms of injury, such as wounds, burns, blunt trauma, and infection. Radiation combined injury would also be expected after a radiological or nuclear attack. Few animal models of radiation combined injury exist, and mechanisms underlying the high mortality associated with complex radiation injuries are poorly understood. Medical countermeasures are currently available for management of the non-radiation components of radiation combined injury, but it is not known whether treatments for other insults will be effective when the injury is combined with radiation exposure. Further research is needed to elucidate mechanisms behind the synergistic lethality of radiation combined injury and to identify targets for medical countermeasures. To address these issues, the National Institute of Allergy and Infectious Diseases convened a workshop to make recommendations on the development of animal models of radiation combined injury, possible mechanisms of radiation combined injury, and future directions for countermeasure research, including target identification and end points to evaluate treatment efficacy.

A summary is provided of presentations and discussions from the NASA Radiation Biomarker Workshop held September 27-28, 2007, at NASA Ames Research Center in Mountain View, California. Invited speakers were distinguished scientists representing key sectors of the radiation research community. Speakers addressed recent developments in the biomarker and biotechnology fields that may provide new opportunities for health-related assessment of radiation-exposed individuals, including for long-duration space travel. Topics discussed include the space radiation environment, biomarkers of radiation sensitivity and individual susceptibility, molecular signatures of low-dose responses, multivariate analysis of gene expression, biomarkers in biodefense, biomarkers in radiation oncology, biomarkers and triage following large-scale radiological incidents, integrated and multiple biomarker approaches, advances in whole-genome tiling arrays, advances in mass-spectrometry proteomics, radiation biodosimetry for estimation of cancer risk in a rat skin model, and confounding factors. Summary conclusions are provided at the end of the report.

This interlaboratory comparison validates the dicentric chromosome assay for assessing radiation dose in mass casualty accidents and identifies the advantages and limitations of an international biodosimetry network. The assay's validity and accuracy were determined among five laboratories following the International Organization for Standardization guidelines. Blood samples irradiated at the Armed Forces Radiobiology Research Institute were shipped to all laboratories, which constructed individual radiation calibration curves and assessed the dose to dose-blinded samples. Each laboratory constructed a dose-effect calibration curve for the yield of dicentrics for (60)Co gamma rays in the 0 to 5-Gy range, using the maximum likelihood linear-quadratic model, Y = c + alphaD + betaD(2). For all laboratories, the estimated coefficients of the fitted curves were within the 99.7% confidence intervals (CIs), but the observed dicentric yields differed. When each laboratory assessed radiation doses to four dose-blinded blood samples by comparing the observed dicentric yield with the laboratory's own calibration curve, the estimates were accurate in all laboratories at all doses. For all laboratories, actual doses were within the 99.75% CI for the assessed dose. Across the dose range, the error in the estimated doses, compared to the physical doses, ranged from 15% underestimation to 15% overestimation.

The health effects of embedded fragments of depleted uranium (DU) are being investigated to determine whether current surgical fragment-removal policies are appropriate for this metal. The authors studied rodents implanted with DU pellets as well as cultured human cells exposed to DU compounds. Results indicate that uranium from implanted DU fragments distributes to tissues distant from implantation sites, including bone, kidney, muscle, and liver. Despite levels of uranium in kidney that would be nephrotoxic after acute exposure, no histological or functional kidney toxicity was observed with embedded DU, indicating that the kidney adapts when exposed chronically. Nonetheless, further studies of the long-term health impact are needed. DU is mutagenic and transforms human osteoblastic cells into a tumorigenic phenotype. It alters neurophysiological parameters in rat hippocampus, crosses the placental barrier, and enters fetal tissue. Preliminary data also indicate decreased rodent litter size when animals are bred 6 months or longer after DU implantation.

During the Persian Gulf War, soldiers were injured with depleted uranium (DU) fragments. To assess the potential health risks associated with chronic exposure to DU, Sprague Dawley rats were surgically implanted with DU pellets at 3 dose levels (low, medium and high). Biologically inert tantalum (Ta) pellets were used as controls. At 1 day and 6, 12, and 18 months after implantation, the rats were euthanized and tissue samples collected. Using kinetic phosphorimetry, uranium levels were measured. As early as 1 day after pellet implantation and at all subsequent sample times, the greatest concentrations of uranium were in the kidney and tibia. At all time points, uranium concentrations in kidney and bone (tibia and skull) were significantly greater in the high-dose rats than in the Ta-control group. By 18 months post-implantation, the uranium concentration in kidney and bone of low-dose animals was significantly different from that in the Ta controls. Significant concentrations of uranium were excreted in the urine throughout the 18 months of the study (224 +/- 32 ng U/ml urine in low-dose rats and 1010 +/- 87 ng U/ml urine in high-dose rats at 12 months). Many other tissues (muscle, spleen, liver, heart, lung, brain, lymph nodes, and testicles) contained significant concentrations of uranium in the implanted animals. From these results, we conclude that kidney and bone are the primary reservoirs for uranium redistributed from intramuscularly embedded fragments. The accumulations in brain, lymph nodes, and testicles suggest the potential for unanticipated physiological consequences of exposure to uranium through this route.

Scientists are just beginning to understand the complicated response to radiation exposure that organisms exhibit. In their Perspective, [ Coleman et al .][1] report on a recent meeting that reviewed the model systems available to study these issues and recent progress in their application to radiation injury. [1]: http://www.sciencemag.org/cgi/content/full/304/5671/693

To understand the neuropathological roles of free radicals we investigate their actions in a model neuronal system, the hippocampal brain slice. Free radicals can be generated through a number of methods: hydrogen peroxide to produce hydroxyl radicals, dihydroxyfumarate to generate superoxide and ionizing radiation producing a variety of radical species. We find that free radicals have a number of profound effects in this system, which can be prevented by free-radical scavengers and antioxidants. With exposure to free radicals, the ability to generate spikes and synaptic efficacy are impaired. Decreased spike generating ability is correlated with lipid peroxidation. No change in membrane potential, membrane resistance, or many of the potassium currents can account for the effect on spike generation. Protein oxidation is likely to underlie synaptic damage. Both inhibitory and excitatory synaptic potentials are reduced by free-radical exposure. Presynaptic mechanisms are implicated. Lower concentrations of radicals prevent the maintenance of long-term potentiation, perhaps through oxidation of the NMDA receptor. The actions of the free radicals are often reversible because of the presence of repair mechanisms, such as glutathione, in hippocampal slices. The brain slice preparation has allowed us to begin to understand the electrophysiological and biochemical consequences of free-radical exposure.

The importance of glial cells in controlling the neuronal microenvironment has been increasingly recognized. We now demonstrate that glial cells play an integral role in hippocampal synaptic transmission by using the glial-specific metabolic blocker fluoroacetate (FAC) to selectively inhibit glial cell function. FAC inhibits evoked intracellular postsynaptic potentials (PSPs; IC50 = 39 μM) as well as population PSPs (IC50 = 65 μM) in field CA1 of the guinea pig hippocampal slice. Spontaneous synaptic transmission is concurrently decreased. These effects are time and dose dependent. ATP concentrations in glial but not neuronal elements are also significantly reduced with FAC treatment. Simultaneous application of the metabolic substrate isocitrate with FAC prevents both the reduction in glial ATP concentrations and the decrease in evoked PSPs. Given that isocitrate is selectively taken up by glia, these data further support a glial specific metabolic action of FAC. Additionally, FAC has no postsynaptic effects as peak responses to iontophoretically applied glutamate are unchanged. However, the decay of both iontophoretic and evoked PSPs are prolonged following FAC treatment suggesting inhibition of glutamate uptake may contribute to the FAC-induced depression of synaptic potentials. These results show, for the first time, that glial cells are critical for maintenance of synaptic transmission and suggest a role for glial cells in the modulation of synaptic efficacy. © 1994 Wiley-Liss, Inc.

Depletion of glutathione (GSH), an intrinsic antioxidant, increases vulnerability to free radical damage in a number of cell systems. This study investigates the role of GSH in limiting electrophysiological damage and/or recovery from free radical exposure in slices of guinea pig hippocampus. Synaptic potentials (PSPs) and population spikes (PSs) were recorded from field CA1. Free radicals were generated from 0.006% peroxide through the Fenton reaction. Analysis of the input-output curves showed that peroxide treatment decreased PSPs and impaired ability of the PSPs to generate PSs as previously reported. Recovery was nearly total within a half hour. Treatment with 5 mM buthionine sulfoximine (BSO) for 2 h depleted hippocampal GSH to 79.2% of control values. The extent of free radical damage was not increased. Recovery, however, was only partial. GSH was further depleted by oxidation with diamide or covalent bonding with dimethyl fumarate (DMF) immediately before and during the peroxide treatment. Neither diamide nor DMF treatment in BSO-incubated tissue enhanced peroxide-induced electrophysiological deficits. Following these treatments, however, tissue showed little recovery from free radical damage. We conclude that glutathione is essential for repair processes in hippocampal neurons exposed to oxidative damage.

Guinea pigs were exposed to 5 and 10 Gy gamma radiation. Hippocampal brain slices were isolated 30 min, 1 day, 3 days and 5 days after irradiation or sham irradiation and the electrophysiological characteristics of the neural tissue were evaluated. Both radiation doses elicited significant changes that were dependent on dose, dose rate and time. Synaptic efficacy decreased soon after exposure to 5 Gy at dose rates of both 1 and 20 Gy/min. Recovery occurred by 5 days. Ten grays at 20 Gy/min potentiated the postsynaptic potential 1 day after irradiation. By 3 days, synaptic efficacy was decreased and did not recover. The ability of the synaptic potentials to generate spikes was potentiated within 30 min after exposure to 5 Gy at 1 Gy/min and persisted through 3 days, with recovery at 5 days. At the 20 Gy/min dose rate, a similar potentiation did not result with 10 Gy and occurred only at 3 days after irradiation with 5 Gy. Rather, within 30 min and after 5 days, spike generation was significantly depressed by these exposures. Both synaptic efficacy and spike generation contribute to the net input-output relationship of the neuronal population. This relationship was profoundly decreased within 30 min with recovery at 1 day and subsequent decline with the higher dose rate in a dose-dependent manner. These persistent changes in neuronal function are likely to be a consequence of the actions of ionizing radiation on the physiological processes that influence the neuronal environment.

A new method of exposing tissues to X rays in a lead Faraday cage has made it possible to examine directly radiation damage to isolated neuronal tissue. Thin slices of hippocampus from brains of euthanized guinea pigs were exposed to 17.4 ke V X radiation. Electrophysiological recordings were made before, during, and after exposure to doses between 5 and 65 Gy at a dose rate of 1.54 Gy/min. Following exposure to doses of 40 Gy and greater, the synaptic potential was enhanced, reaching a steady level soon after exposure. The ability of the synaptic potential to generate a spike was reduced and damage progressed after termination of the radiation exposure. Recovery was not observed following termination of exposure. These results demonstrate that an isolated neuronal network can show complex changes in electrophysiological properties following moderate doses of ionizing radiation. An investigation of radiation damage directly to neurons in vitro will contribute to the understanding of the underlying mechanisms of radiation-induced nervous system dysfunction.

Intracellular pressure injection of cyclic AMP induces a slow voltage-dependent inward current in some neurons of Aplysia californica. The time course, voltage dependence, and ionic sensitivities of this response are nearly identical to those of the voltage-dependent calcium current induced by serotonin in the same preparation. The response to cyclic AMP is unaffected by changes in the extracellular concentration of chloride or potassium. The current is slowly but minimally reduced by a sodium-free solution. The calcium channel blocker, cadmium, blocks the current elicited by injection of cyclic AMP. The data presented here suggest that cyclic AMP can induce a voltage-dependent calcium current.

The effects of furosemide on action potentials and responses to several neurotransmitters have been studied in the neurons of Aplysia. Furosemide (10(-7) and 10(-3) M) does not visibly affect the normal action potential in R15 neurons. However, when TTX (30 microM) is used to block the sodium component in R15, the remaining spike (presumably the calcium component) is increased in amplitude in the presence of furosemide. Furosemide also alters transmitter-induced conductances. Furosemide greatly reduces the amplitude and shifts, in a depolarizing direction, the reversal potential of chloride-dependent responses to gamma-aminobutyric acid (GABA) and acetylcholine (ACh). This suggests that furosemide both blocks the chloride channel and inhibits a chloride pump. ACh-induced sodium responses were also reduced by furosemide but to a lesser extent than chloride responses. The potassium response to ACh and a voltage-dependent calcium response to serotonin were not altered. These results indicate that furosemide could alter synaptic responses both presynaptically by enhancement of calcium flux during the action potential and postsynaptically by blockade of chloride and sodium conductances.

The radioprotectant, dithiothreitol (DTT) has been shown to increase excitability in the hippocampal slice preparation. In the present study, intracellular recording techniques were used to further examine the actions of DTT. Electrophysiological recordings from CA1 pyramidal cells were obtained prior to, during and after DTT exposure. DTT caused a small depolarization without altering membrane resistance. DTT induced spontaneous firing and occasional burst firing in normally silent neurons. These effects were accompanied by a reduction in spike frequency adaptation but no change in the afterhyperpolarization following a train of action potentials. Following DTT exposure, orthodromic stimulation produced multiple firing. Subthreshold excitatory postsynaptic potentials (EPSPs) were significantly prolonged. Isolating the CA1 subfield, attenuated the prolongation of the EPSP by DTT. Recurrent inhibitory postsynaptic potentials were unaffected by DTT. The actions of DTT are likely to result from DTT-induced reduction of disulfide bonds since the reduced form of DTT does not cause a similar hyperexcitability.

A 50 kVp molybdenum target/filter x-ray tube has been installed inside a lead-shielded Faraday cage. High-dose rates of up to 1.54 Gy min −1 (17.4 keV weighted average photons) have been used to conduct local in vitro irradiations of the hippocampal region of guinea pig brains. Electrophysiological recordings of subtle changes in neuronal activity indicate this system is suitable for this application.

Free radicals and active oxygen compounds are implicated in brain ischemia and head trauma. Previous studies have shown that free radicals, generated by radiation and through the Fenton reaction, produce both synaptic and postsynaptic damage in the hippocampal brain slice. 1,2,3 To evaluate the contribution of oxidation to the observed damage the actions of the oxidants, chloramine-T and N-chlorosuccinimide (NCS), were studied on electrophysiological responses in the hippocampal slice isolated from the brains of guinea pigs. Electrical stimulation of afferents to neurons of the CAI region of hippocampus evoked a population postsynaptic potential (population PSP) in the dendritic layer and a population spike in the cell body layer. Chloramine-T (25–500 μM) and NCS (750–4000 μM) decreased the population spike in a dose-dependent manner ( ED 50 ⋍ 125 μ M and 1100 μM, respectively). Input/output curves revealed that both the population spike and the population PSP were significantly reduced with both oxidants; but, the ability of the population PSP to produce a population spike was not impaired. These studies suggest that oxidation reactions can account for the synaptic component of the damage produced by free radicals but can not account for the postsynaptic effects.

To study the effects of active oxygen on neuronal electrophysiology, hippocampal brain slices were exposed to hydrogen peroxide plus ferrous sulfate which react to produce hydroxyl free radicals. Analysis of extracellularly recorded somatic and dendritic responses to orthodromic stimulation indicated a decrease in both synaptic efficacy and impairment of action potential generation.

Dithiothreitol (DTT) is a sulfhydryl reducing agent used as a radioprotectant. Exposure of hippocampal slices for 30 min to 0.5 mM DTT irreversibly increased the orthodromic population spike amplitude, promoted repetitive firing and induced spontaneous epileptiform activity in the CA1 subfield. The same concentration of the oxidized form of DTT did not increase hippocampal excitability. Although the slope of the population synaptic response to afferent stimulation (popPSP) was unchanged by DTT, the duration of the popPSP was prolonged. Recurrent inhibition was unaffected. DTT probably exerts its effects through an irreversible chemical reaction with cellular components. Possible mechanisms of DTT-induced epileptiform activity are discussed.

The afterhyperpolarization (AHP) that follows repetitive stimulation was recorded intracellularly from CA1 pyramidal neurons in the guinea pig hippocampal slice preparation. Although the late AHP could be blocked by histamine (1-10 microM), forskolin (10 microM) and 8-bromo-cyclic AMP (100 and 500 microM), neither prostaglandins D2, E1 and F2 alpha (0.5 microM) nor vasoactive intestinal polypeptide (0.5 microM) had any effect on the AHP, membrane potential, membrane resistance or action potential properties.

A voltage-dependent inward current is elicited by intracellular application of cyclic AMP in some neurons of Aplysia. The current has a slow time course and is present only at potentials more depolarized than −30 mV. Two other methods of increasing cellular cyclic AMP (phosphodiesterase inhibitors and sodium fluoride activation of adenylate cyclase) also induce an inward current at depolarized potentials.

Octopamine and dopamine evoke a slow voltage-dependent inward current in neurons of Aplysia. The responses are present only at depolarized potentials (−40 mV). They are insensitive to changes in potassium, persist after removal of sodium, are not reduced by low chloride solution, and are blocked by cadmium. Because of the similarities in voltage-dependence, time course and ionic sensitivities, the ionic mechanism of the voltage-dependent responses to octopamine and dopamine are concluded to be the same as that of the voltage-dependent response to serotonin reported previously 13.