Models for evaluating agents intended for the prophylaxis, mitigation and treatment of radiation injuries. Report of an NCI Workshop, December 3-4, 2003

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.     

Molecular and cellular biology of moderate-dose (1-10 Gy) radiation and potential mechanisms of radiation protection: report of a workshop at Bethesda,Maryland, December 17-18, 2001

Exposures to doses of radiation of 1-10 Gy, defined in this workshop as moderate-dose radiation, may occur during the course of radiation therapy or as the result of radiation accidents or nuclear/radiological terrorism alone or in conjunction with bioterrorism. The resulting radiation injuries would be due to a series of molecular, cellular, tissue and whole-animal processes. To address the status of research on these issues, a broad-based workshop was convened. The specific recommendations were: (1) Research: Identify the key molecular, cellular and tissue pathways that lead from the initial molecular lesions to immediate and delayed injury. The latter is a chronic progressive process for which postexposure treatment may be possible. (2) Technology: Develop high-throughput technology for studying gene, protein and other biochemical expression after radiation exposure, and cytogenetic markers of radiation exposure employing rapid and accurate techniques for analyzing multiple samples. (3) Treatment strategies: Identify additional biological targets and develop effective treatments for radiation injury. (4) Ensuring sufficient expertise: Recruit and train investigators from such fields as radiation biology, cancer biology, molecular biology, cellular biology and wound healing, and encourage collaboration on interdisciplinary research on the mechanisms and treatment of radiation injury. Communicate knowledge of the effects of radiation exposure to the general public and to investigators, policy makers and agencies involved in response to nuclear accidents/events and protection/treatment of the general public.     

Efficacy of radiation countermeasures depends on radiation quality

The detonation of a nuclear weapon or a nuclear accident represent possible events with significant exposure to mixed neutron/γ-radiation fields. Although radiation countermeasures generally have been studied in subjects exposed to pure photons (γ or X rays), the mechanisms of injury of these low linear energy transfer (LET) radiations are different from those of high-LET radiation such as neutrons, and these differences may affect countermeasure efficacy. We compared 30-day survival in mice after varying doses of pure γ and mixed neutron/γ (mixed field) radiation (MF, Dn/Dt = 0.65), and also examined peripheral blood cells, bone marrow cell reconstitution, and cytokine expression. Mixed-field-irradiated mice displayed prolonged defects in T-cell populations compared to mice irradiated with pure γ photons. In mouse survival assays, the growth factor granulocyte colony-stimulating factor (G-CSF) was effective as a (post-irradiation) mitigator against both γ-photons and mixed-field radiation, while the thrombopoietin (TPO) mimetic ALXN4100TPO was effective only against γ irradiation. The results indicate that radiation countermeasures should be tested against radiation qualities appropriate for specific scenarios before inclusion in response plans.     

Animal models for radiation injury, protection and therapy

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.     

Progress in research on radiation combined injury in China

The significant feature of radiation combined injury is the occurrence of a combined effect. For decades our institute has focused on studying the key complications of radiation-burn injury, including shock, suppression of hematopoiesis and immunity, gastrointestinal damage and local refractory wound healing. Here we summarize recent advancements in elucidating the mechanisms of and potential treatments for radiation combined injury. Concerning the suppression and regeneration of hematopoiesis in radiation combined injury, mechanisms of megakaryocyte damage have been elucidated and a new type of fusion protein stimulating thrombopoiesis has been developed and is being tested in animals. With regard to the damage and repair of intestinal epithelium, the important molecular mechanisms of radiation combined injury have been clarified, and new measures to prevent and treat gastrointestinal tract injury are proposed. With respect to the difficulties encountered in wound healing, the underlying causes of radiation combined injury have been proposed, and some potential methods to accelerate wound closure are under study. Systemic experiments have been done to determine the appropriate time for eschar excision and skin grafting, and the results provided significant insight into clinical treatment of the injury. In the search for early therapeutic regimens for severe burns and radiation combined injury to prevent deterioration of injuries and to improve survival, cervical sympathetic ganglion block was used for the treatment of animals with radiation combined injury and had significant benefits. These research advancements have potential for application in on-site emergency rescue and in-hospital treatment of radiation combined injury.     

Hypersensitivity to very-low single radiation doses: Its relationship to the adaptive response and induced radioresistance

There is now little doubt of the existence of radioprotective mechanisms, or stress responses, that are upregulated in response to exposure to small doses of ionizing radiation and other DNA-damaging agents. Phenomenologically, there are two ways in which these induced mechanisms operate. First, a small conditioning dose (generally below 30 cGy) may protect against a subsequent, separate, exposure to radiation that may be substantially larger than the initial dose. This has been termed the adaptive response. Second, the response to single doses may itself be dose-dependent so that small acute radiation exposures, or exposures at very low dose rates, are more effective per unit dose than larger exposures above the threshold where the induced radioprotection is triggered. This combination has been termed low-dose hypersensitivity (HRS) and induced radioresistance (IRR) as the dose increases. Both the adaptive response and HRS/IRR have been well documented in studies with yeast, bacteria, protozoa, algae, higher plant cells, insect cells, mammalian and human cells in vitro, and in studies on animal models in vivo. There is indirect evidence that the HRS/IRR phenomenon in response to single doses is a manifestation of the same underlying mechanism that determines the adaptive response in the two-dose case and that it can be triggered by high and low LET radiations as well as a variety of other stress-inducing agents such as hydrogen peroxide and chemotherapeutic agents although exact homology remains to be tested. Little is currently known about the precise nature of this underlying mechanism, but there is evidence that it operates by increasing the amount and rate of DNA repair, rather than by indirect mechanisms such as modulation of cell-cycle progression or apoptosis. Changed expression of some genes, only in response to low and not high doses, may occur within a few hours of irradiation and this would be rapid enough to explain the phenomenon of induced radioresistance although its specific molecular components have yet to be identified.     

Experimental research on the management of combined radiation-burn injury in China

Combined radiation-burn injury can occur in people exposed to nuclear explosions, nuclear accidents or radiological terrorist attacks. Using different combined radiation-burn injury animal models, the pathological mechanisms underlying combined radiation-burn injury and effective medical countermeasures have been explored for several years in China, mainly at our institute. Targeting key features of combined radiation-burn injury, several countermeasures have been developed. Fluid transfusion and the calcium antagonist verapamil can prevent early shock and improve myocardial function after combined radiation-burn injury. Recombinant human interleukin 4 (rhIL-4) is able to effectively reduce bacterial infection and increase intestinal immunological ability. Chitosan-wrapped human defensin 5 (HD5) and glucagon-like peptide 2 (GLP-2) nanoparticles can increase the average survival time of animals with severe combined radiation-burn injury. After treatment by cervical sympathetic ganglia block (SB), hematopoietic function is promoted and the release of inflammatory cytokines is suppressed. The optimal time for escharectomy and allo-skin grafting is 24 h after injury. Transfusion of irradiated (20 Gy) or stored (4°C, 7 days) blood improves the survival of allo-skin grafting and allo-bone marrow cells. In conclusion, as our understanding of the mechanisms of combined radiation-burn injury has progressed, new countermeasures have been developed for its treatment. Because of the complexity of its pathology and the difficulty in clinical management, further efforts are needed to improve the treatment of this kind of injury.     

Priority list of research areas for radiological nuclear threat countermeasures

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.     

Pathophysiological Responses in Rat and Mouse Models of Radiation-Induced Brain Injury

The brain is the major dose-limiting organ in patients undergoing radiotherapy for assorted conditions. Radiation-induced brain injury is common and mainly occurs in patients receiving radiotherapy for malignant head and neck tumors, arteriovenous malformations, or lung cancer-derived brain metastases. Nevertheless, the underlying mechanisms of radiation-induced brain injury are largely unknown. Although many treatment strategies are employed for affected individuals, the effects remain suboptimal. Accordingly, animal models are extremely important for elucidating pathogenic radiation-associated mechanisms and for developing more efficacious therapies. So far, models employing various animal species with different radiation dosages and fractions have been introduced to investigate the prevention, mechanisms, early detection, and management of radiation-induced brain injury. However, these models all have limitations, and none are widely accepted. This review summarizes the animal models currently set forth for studies of radiation-induced brain injury, especially rat and mouse, as well as radiation dosages, dose fractionation, and secondary pathophysiological responses.     

Using genetically engineered mice for radiation research

The laboratory mouse has been used for many decades as a model system for radiation research. Recent advances in genetic engineering now allow scientists to delete genes in specific cell types at different stages of development. The ability to manipulate genes in the mouse with spatial and temporal control opens new opportunities to investigate the role of genes in regulating the response of normal tissues and tumors to radiation. Currently, we are using the Cre-loxP system to delete genes, such as p53, in a cell-type specific manner in mice to study mechanisms of acute radiation injury and late effects of radiation. Our results demonstrate that p53 is required in the gastrointestinal (GI) epithelium to prevent radiation-induced GI syndrome and in endothelial and/or hematopoietic cells to prevent late effects of radiation. We have also used these genetic tools to generate primary tumors in mice to study tumor response to radiation therapy. These advances in genetic engineering provide a powerful model system to dissect both the mechanisms of normal tissue injury after irradiation and the mechanisms by which radiation cures cancer.     

Iron-ion radiation accelerates atherosclerosis in apolipoprotein E-deficient mice

Radiation exposure from a number of terrestrial sources is associated with an increased risk for atherosclerosis. Recently, concern over whether exposure to cosmic radiation might pose a similar risk for astronauts has increased. To address this question, we examined the effect of 2 to 5 Gy iron ions ((56)Fe), a particularly damaging component of cosmic radiation, targeted to specific arterial sites in male apolipoprotein E-deficient (apoE(-/-)) mice. Radiation accelerated the development of atherosclerosis in irradiated portions of the aorta independent of any systemic effects on plasma lipid profiles or circulating leukocytes. Further, radiation exposure resulted in a more rapid progression of advanced aortic root lesions, characterized by larger necrotic cores associated with greater numbers of apoptotic macrophages and reduced lesional collagen compared to sham-treated mice. Intima media thickening of the carotid arteries was also exacerbated. Exposure to (56)Fe ions can therefore accelerate the development of atherosclerotic lesions and promote their progression to an advanced stage characterized by compositional changes indicative of increased thrombogenicity and instability. We conclude that the potential consequences of radiation exposure for astronauts on prolonged deep-space missions are a major concern. Knowledge gained from further studies with animal models should lead to a better understanding of the pathophysiological effects of accelerated ion radiation to better estimate atherogenic risk and develop appropriate countermeasures to mitigate its damaging effects.     

In vitro cultured cells as probes for space radiation effects on biological systems

Near future scenarios of long-term and far-reaching manned space missions, require more extensive knowledge of all possible biological consequences of space radiation, particularly in humans, on both a long-term and a short-term basis. In vitro cultured cells have significantly contributed to the tremendous advancement of biomedical research. It is therefore to be expected that simple biological systems such as cultured cells, will contribute to space biomedical sciences. Space represents a novel environment, to which life has not been previously exposed. Both microgravity and space radiation are the two relevant components of such an environment, but biological adaptive mechanisms and efficient countermeasures can significantly minimize microgravity effects. On the other hand, it is felt that space radiation risks may be more relevant and that defensive strategies can only stem from our deeper knowledge of biological effects and of cellular repair mechanisms. Cultured cells may play a key role in such studies. Particularly, thyroid cells may be relevant because of the exquisite sensitivity of the thyroid gland to radiation. In addition, a clone of differentiated, normal thyroid follicular cells (FRTL5 cells) is available in culture, which is well characterized and particularly fit for space research.     

Combined effects of gamma radiation and arsenite on the proteome of human TK6 lymphoblastoid cells

Arsenic present in drinking water and mining environments in some areas has been associated with an increased rate of skin and internal cancers. Contrary to the epidemiological evidence in humans, arsenic does not induce cancer in animal models, but is able to enhance the mutagenicity of other agents. In order to achieve a better understanding of the interaction between arsenic and ionising radiation, an investigation was conducted to detect differences at the proteome level of human TK6 lymphoblastoid cells exposed to these agents. Cells were exposed to either a single dose of 1-Gy 137Cs-gamma-rays or to 1 microM arsenite (As(III)) or to both agents in combination. Two-dimensional (2D) electrophoresis and matrix-assisted laser desorption/ionisation-time of flight (MALDI-TOF) were employed for the screening and identification of proteins, respectively. It proved possible to identify seven proteins with significantly affected abundance, three of which showed increased levels and the remaining four showed decreased levels under at least one of the exposure conditions. Following arsenite treatment or irradiation, a significant increase compared with that of the control was observed for glutathione (GSH) transferase omega 1 and proteasome subunit beta type 4 precursor. The combined exposure did not result in an induction of the enzymes. The expression of electron-transfer flavoprotein subunit alpha was found to be enhanced under all three-exposure conditions. Ubiquinol-cytochrome C reductase complex core protein I, adenine phosphoribosyl transferase and endoplasmic reticulum protein hERp29 showed decreased levels after irradiation or arsenite treatment, but not after the combined exposure. The level of serine/threonine protein phosphatase 1 alpha decreased with all treatments. The main conclusions are that both arsenite and gamma-radiation influence the levels of several proteins involved in major metabolic and regulatory pathways, either directly or by triggering the defence mechanisms of the cell. The combined effect of both exposures on the level of some essential proteins such as glutathione transferase, proteasome or serine/threonine phosphatase may contribute to the co-carcinogenic effect of arsenic.     

Ultraviolet radiation,resistance to infectious diseases,and vaccination responses

Exposure to ultraviolet (UV) radiation, as in sunlight, can modulate immune responses in animals and humans. This immunomodulation can lead to positive health effects especially with respect to certain autoimmune diseases and allergies. However, UV-induced immunomodulation has also been shown to be deleterious. Experimental animal studies have revealed that UV exposure can impair resistance to many infectious agents, such as bacteria, parasites, viruses, and fungi. Importantly, these effects are not restricted to skin-associated infections, but also concern systemic infections. The real consequences of UV-induced immunomodulation on resistance to infectious diseases are not known for humans. Risk estimations have been performed through extrapolation of animal data, obtained from infection models, to the human situation. This estimation indicated that UV doses relevant to outdoor exposure can impair the human immune system sufficiently to have effects on resistance to infections. To further quantify and validate this risk estimation, data, e.g., from human volunteer studies, are necessary. Infection models in humans are not allowed for ethical reasons. However, vaccination against an infectious disease evokes a similar immune response as the pathogen and thereby provides an opportunity to measure the effect of UV radiation on the immune system and an estimate of the possible consequences of altered resistance to infectious agents. Effects of controlled UVB exposure on immune responses after hepatitis B vaccination have been established in mice and human volunteers. In mice, cellular and Th1-associated humoral immune responses to hepatitis B were significantly impaired, whereas in human volunteers no significant effect of UVB on these responses could be found. Preliminary data indicate that cytokine polymorphisms might be, at least in part, responsible for interindividual differences in immune responses and in susceptibility to UVB-induced immunomodulation. In addition, adaptation to UV exposure needs to be considered as a possible explanation for the difference between mice and humans that was observed in the hepatitis B vaccination model.     

In vivo spin trapping of free radicals generated in brain, spleen, and liver during gamma radiation of mice

Spin trapping techniques combined with electron spin resonance spectroscopy were used to capture and detect free radicals generated in vivo during exposure to ionizing radiation. Tissue extracts of mice given an intraperitoneal or intragastric dose of the spin trap, alpha-phenyl-t-butyl nitrone prior to exposure to gamma radiation (2 to 5 Gy), contained a radical adduct with hyperfine splitting constants characteristic of spin adducts of carbon-centered lipid radicals. Considerably more radicals were trapped in tissues when the trap was given 3 h before radiation as compared to 30 min before exposure. The radicals observed may either be secondary species resulting from an attack on cellular components by products of water radiolysis, or primary radicals resulting from direct interaction of the radiation with biological molecules. The results indicate that the spin trapping agent is able to penetrate well into animal tissues, and to capture radical species under conditions where the latter would be expected to occur.     

Emphasis of biological research for space radiation]

The paper summarized issues, current status and the recent topics in biological research of space radiation. Researches to estimate a risk associated with space radiation exposure during a long-term manned space flight, such as in the International Space Station, is emphasized because of the large uncertainty of biological effects and a complexity of the radiation environment in space. The Issues addressed are; 1) biological effects and end points in low dose radiation, 2) biological effects under low dose rate and long-term radiation exposure, 3) modification of biological responses to radiation under space environments, 4) various aspects of biological end points vs. cellular and molecular mechanisms, 5) estimation of human risk associated with radiation exposure in space flight, 6) regulations for radiation exposure limits for space workers. The paper also summarized and introduced recent progress in space related radiation researches with various biological systems.     

Cancer risk from chronic exposures to chemicals and radiation: a comparison of the toxicological reference value with the radiation detriment

This article aims at comparing reference methods for the assessment of cancer risk from exposure to genotoxic carcinogen chemical substances and to ionizing radiation. For chemicals, cancer potency is expressed as a toxicological reference value (TRV) based on the most sensitive type of cancer generally observed in animal experiments of oral or inhalation exposure. A dose–response curve is established by modelling experimental data adjusted to apply to human exposure. This leads to a point of departure from which the TRV is derived as the slope of a linear extrapolation to zero dose. Human lifetime cancer risk can then be assessed as the product of dose by TRV and it is generally considered to be tolerable in a 10^–6–10^–4 range for the public in a normal situation. Radiation exposure is assessed as an effective dose corresponding to a weighted average of energy deposition in body organs. Cancer risk models were derived from the epidemiological follow-up of atomic bombing survivors. Considering a linear-no-threshold dose-risk relationship and average baseline risks, lifetime nominal risk coefficients were established for 13 types of cancers. Those are adjusted according to the severity of each cancer type and combined into an overall indicator denominated radiation detriment. Exposure to radiation is subject to dose limits proscribing unacceptable health detriment. The differences between chemical and radiological cancer risk assessments are discussed and concern data sources, extrapolation to low doses, definition of dose, considered health effects and level of conservatism. These differences should not be an insuperable impediment to the comparison of TRVs with radiation risk, thus opportunities exist to bring closer the two types of risk assessment.

     

Experimental radiation-induced heart disease: past, present, and future

Radiation-induced heart disease (RIHD) is a serious side effect of radiotherapy for intrathoracic and chest wall tumors. The threshold dose for development of clinically significant RIHD is believed to be lower than previously assumed. Therefore, research into mechanisms of RIHD has gained substantial momentum. RIHD becomes clinically apparent ten to fifteen years after radiation exposure. Chronic manifestations of RIHD include accelerated atherosclerosis, cardiomyopathy, and valve abnormalities. Reducing exposure of the heart during radiotherapy is the only known method of preventing RIHD, and there are no approaches to reverse RIHD once it occurs. We use a combination of pharmacological and genetic animal models to determine biological mechanisms of RIHD. Major technological advances in small animal research have made this type of study more valuable. The long-term goal of this work is to identify targets for intervention in RIHD, thereby enhancing the efficacy and safety of thoracic radiotherapy.     

Altitude, radiation, and mortality from cancer and heart disease

The variation in background radiation levels is an important source of information for estimating human risks associated with low-level exposure to ionizing radiation. Several studies conducted in the United States, correlating mortality rates for cancer with estimated background radiation levels, found an unexpected inverse relationship. Such results have been interpreted as suggesting that low levels of ionizing radiation may actually confer some benefit. An environmental factor strongly correlated with background radiation is altitude. Since there are important physiological adaptations associated with breathing thinner air, such changes may themselves influence risk. We therefore fit models that simultaneously incorporated altitude and background radiation as predictors of mortality. The negative correlations with background radiation seen for mortality from arteriosclerotic heart disease and cancers of the lung, the intestine, and the breast disappeared or became positive once altitude was included in the models. By contrast, the significant negative correlations with altitude persisted with adjustment for radiation. Interpretation of these results is problematic, but recent evidence implicating reactive forms of oxygen in carcinogenesis and atherosclerosis may be relevant. We conclude that the cancer correlational studies carried out in the United States using vital statistics data do not in themselves demonstrate a lack of carcinogenic effect of low radiation levels, and that reduced oxygen pressure of inspired air may be protective against certain causes of death.     

Failla memorial lecture. Risk, research, and radiation protection

Radiation protection concerns the risk of stochastic late effects, especially cancer, and limits on radiation exposure both occupationally and for the public tend to be based on these risks. The risks are determined, mainly by expert committees, from the steadily growing information on exposed human populations, especially the survivors of the atomic bombs dropped in Japan in 1945. Risks of cancer estimated up to the early 1980s were in the range 1 to 5 X 10(-2)/Sv, but recent revisions in the dosimetry of the Japanese survivors and additional cycles of epidemiological information suggest values now probably at the high end of this range. These are likely to require an increase in the values used for radiation protection. A major problem with risk estimation is that data are available only for substantial doses and must be extrapolated down to the low-dose region of interest in radiation protection. Thus the shape of the dose-response curve is important, and here we must turn to laboratory research. Of importance are studies involving (1) dose rate, which affects the response to low-LET radiation and often to high-LET radiation as well; (2) radiation quality, since the shapes of the dose-response curves for high- and low-LET radiation differ and thus the RBE, the ratio between them, varies, reaching a maximum value RBEM at low doses; and (3) modifiers of the carcinogenic response, which either enhance or reduce the effect of a given dose. Radiation protection depends both on risk information, and especially also on comparisons with other occupational and public risks, and on research, not only for extrapolations of risk to low doses but also in areas where human information is lacking such as in the effects of radiation quality and in modifications of response.