Biological effects of space radiation and development of effective countermeasures

Contents lists available at Life Sciences in Space Research Biological effects of space radiation and development of effectivecountermeasures Department of Radiation Oncology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104-6072, United States Article history: As part of a program to assess the adverse biological effects expected from astronauts' exposure to space Received 21 January 2014 radiation, numerous different biological effects relating to astronauts' health have been evaluated. There Received in revised form 6 February 2014 has been major focus recently on the assessment of risks related to exposure to solar particle event (SPE) Accepted 6 February 2014 radiation. The effects related to various types of space radiation exposure that have been evaluatedare: gene expression changes (primarily associated with programmed cell death and extracellular matrix (ECM) remodeling), oxidative stress, gastrointestinal tract bacterial translocation and immune Solar particle event system activation, peripheral hematopoietic cell counts, emesis, blood coagulation, skin, behavior/fatigue (including social exploration, submaximal exercise treadmill and spontaneous locomotor activity), Acute effects of radiation heart functions, alterations in biological endpoints related to astronauts' vision problems (lumbar Long-term effects of radiation puncture/intracranial pressure, ocular ultrasound and histopathology studies), and survival, as well aslong-term effects such as cancer and cataract development. A number of different countermeasures havebeen identiﬁed that can potentially mitigate or prevent the adverse biological effects resulting fromexposure to space radiation.
2014 The Committee on Space Research (COSPAR). Published by Elsevier Ltd. All rights reserved.
Changes in hematopoietic cell counts after proton or conventional reference radiation exposures . . . . . . . . . . . . . .
Changes in hematopoietic cell counts in mice after irradiation . . . . . . . . . . . . . . . . . . . . . . .
Changes in peripheral leukocyte counts in ferrets after irradiation . . . . . . . . . . . . . . . . . . . . . .
Changes in peripheral leukocyte counts in Yucatan minipigs after irradiation . . . . . . . . . . . . . . . . . .
Summary of effects of SPE radiation on hematopoietic blood cell counts in ferrets, mice and pigs . . . . . . . . . . .
Effect of SPE-like radiation on gastrointestinal tract integrity . . . . . . . . . . . . . . . . . . . . . . . .
Effect of SPE radiation and hindlimb suspension on immune function measured by bacterial challenge . . . . . . . . .
Summary of the effects of SPE radiation on the immune system . . . . . . . . . . . . . . . . . . . . . . .
Abbreviations: ARS, acute radiation sickness; aPTT, activated partial thromboplastin time; BBI, Bowman–Birk inhibitor; BBIC, BBI concentrate; BFO, blood forming or- gans; BK, bradykinin; CNS, central nerve system; CT, computed tomography; DCF, dichloroﬂuorescein; DIC, disseminated intravascular coagulation; DMF, dose modifyingfactor; DNA-PKcs, DNA-dependent protein kinases; DTH, delayed type hypersensitivity; ECM, extracellular matrix; EGb76, quercetin; eSPE, simulated electron SPE; EVA, extra-vehicular activity; GI, gastrointestinal; GCR, galactic cosmic rays; G-CSF, granulocyte colony-stimulating factor; HDR, high dose rate; HS, hindlimb suspension; HZE particles,highly energetic, heavy, charged particles; ICRP, International Commission of Radiation Protection; IFN-α, interferon-alpha; INR, the patient's ‘test' PT value divided by thelaboratory ‘normal' PT value, raised to the power of the International Sensitivity Index; ISS, International Space Station; LAD, left anterior descending; LBP, lipopolysaccha-ride binding protein; LD50, dose expected to kill 50% of the treated subjects; LDR, low dose rate; LET, linear energy transfer; LPS, lipopolysaccharide; MnSOD, manganesesuperoxide dismutase; NAC, N-acetyl cysteine; NASA, National Aeronautics and Space Administration; NCRP, National Council on Radiation Protection and Measurements; NK,Natural Killer; PAMP, pathogen associated molecular patterns; PBMNC, peripheral blood mononuclear cell; PBS, phosphate buffered saline; PHA, phytohemagglutinin; PMN,polymorphonuclear leukocyte; pSPE, simulated proton SPE; PT, prothrombin time; PWS, partial weight suspension; RBE, relative biological effectiveness; SCR, solar cosmicradiation; SEB, surrogate endpoint biomarker; SeM, L-selenomethionine; SOBP, spread out Bragg peak; SPE, solar particle event; SWT, SI–Wu–Tang; TAS, total antioxidantstatus; TBI, total body irradiation; TF, tissue factor; vWF, von Willenbrand factor; WBC, white blood cell.
2214-5524/ 2014 The Committee on Space Research (COSPAR). Published by Elsevier Ltd. All rights reserved.
A.R. Kennedy / Life Sciences in Space Research 1 (2014) 10–43 Effects of radiation on blood coagulation and the development of disseminated intravascular coagulation . . . . . . . . . . .
Increased intracranial pressure and effects potentially related to vision abnormalities evaluated in Yucatan mini-pigs . . . . . . .
Histopathology changes in the eyes of pigs exposed to simulated proton and electron SPE radiation which could be related tovision problems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Ocular ultrasound results of the eyes of the pigs exposed to SPE radiation . . . . . . . . . . . . . . . . . . .
Opening pressure in pigs exposed to proton and electron SPE radiation . . . . . . . . . . . . . . . . . . . .
Countermeasures and mitigation of space radiation damage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Radiation induced oxidative stress and antioxidants as countermeasures . . . . . . . . . . . . . . . . . . . . . . .
Antioxidant protection against radiation induced cell death and transformation in vitro . . . . . . . . . . . . . . . . . .
Antioxidant protection against space radiation induced mortality . . . . . . . . . . . . . . . . . . . . . . . . .
Antioxidant protection against space radiation induced cataracts . . . . . . . . . . . . . . . . . . . . . . . . . .
Antioxidant prevention of space radiation induced cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Mechanism(s) for antioxidants as radiation countermeasures . . . . . . . . . . . . . . . . . . . . . . . . . . .
Granulocyte colony-stimulating factors as countermeasures . . . . . . . . . . . . . . . . . . . . . . . . . . . .
SI–Wu–Tang or fructose as countermeasures to increase neutrophil counts in mice exposed to SPE or γ -ray radiation . . . . . .
Antibiotics as countermeasures for bacterial toxicity in mice exposed to SPE radiation and HS . . . . . . . . . . . . . . .
Countermeasures for radiation induced emesis in ferrets . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Countermeasures for altered bleeding times in ferrets after SPE radiation exposure . . . . . . . . . . . . . . . . . . .
Corticosteroid as a countermeasure for radiation induced pneumonitis and pneumonopathy in pigs . . . . . . . . . . . . .
Mometasone as a countermeasure for SPE proton radiation induced skin lesions in pigs . . . . . . . . . . . . . . . . .
Transparent ﬁlm dressing for protection against proton therapy induced skin reactions in humans . . . . . . . . . . . . .
Summary concerning the major effects of SPE and space radiation . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Changes potentially related to the development of vision abnormalities . . . . . . . . . . . . . . . . . . . . . . .
Threshold radiation doses for statistically signiﬁcant adverse biological effects from SPE-like radiation in vivo . . . . . . . . . .
Agents identiﬁed as countermeasures for space radiation induced adverse biological effects . . . . . . . . . . . . . . . . . .
posed predominately of protons, with a minor contribution fromhelium ions (∼10%) and an even smaller contribution from heavy As reviewed by the pri- ions and electrons (∼1%). SPEs are unpredictable, develop rapidly mary components of radiation in interplanetary space are galactic and usually last for no more than several hours, although some cosmic rays (GCR) and solar cosmic radiation (SCR). GCR originates SPEs may continue for several days. Since protons are the major from outside of our Solar System and consists of 98% baryons component of SPE radiation, ground-based SPE radiation research and 2% electrons. The baryonic component consists of 87% pro- is focused on the biological consequences of proton radiation at tons (hydrogen nuclei), 12% alpha particles (helium nuclei) and the appropriate energies, doses, and dose-rates expected during an approximately 1% of heavier nuclei with atomic numbers up to SPE. A large fraction of the protons during an SPE are in the range 92 (uranium). These heavier nuclei include highly energetic, heavy, of around 50 MeV, but there are also varying levels of protons charged particles known as HZE particles. Although 56Fe ions, as a of higher energies characterizing each individual SPE speciﬁc type of HZE particle, account for less than 1% of the GCR particle ﬂuxes, they contribute signiﬁcantly to the total radiation Exposure to space radiation may place astronauts at signiﬁ- dose received by individual cells exposed to GCR due to the fact cant risk for acute radiation sickness (ARS), signiﬁcant skin injury that the dose to an individual cell is proportional to the square of and numerous other biological effects resulting from exposure to the particle's energy dependent effective charge radiation from a major SPE, which normally includes some HZE SCR consists of low energy solar wind particles that ﬂow con- particles, or combined SPE and GCR. Doses absorbed by tissues stantly from the Sun and the highly energetic solar particle events vary for different SPEs and model systems have been developed (SPEs) that originate from magnetically disturbed regions of the to calculate the radiation doses that could have been received by Sun, which sporadically emit bursts of energetic charged parti- astronauts during previous SPEs For instance, it has been estimated that the August 1972 SPE could have delivered A.R. Kennedy / Life Sciences in Space Research 1 (2014) 10–43 doses of approximately 2.69 Gy and 0.46 Gy to skin and blood ation Exposure, Risk of Acute Radiation Syndromes Due to Solar forming organs (BFO), respectively, in a spacecraft and 32 Gy and Particle Events (SPEs), Risk of Degenerative Tissue or other Health 1.38 Gy to skin and BFO, respectively, during extra-vehicular ac- Effects from Radiation Exposure, and Risk of Radiation Carcino- tivity (EVA). Depending on the radiation dose, dose rate and qual- genesis. The Degenerative Tissue Risks include adverse radiation ity, exposure to radiation during space missions may immediately biologic effects on the heart, circulatory, endocrine, digestive, lens affect the probability for successful mission completion (mission and other tissue systems (which would include radiation effects on critical) or result in late radiation effects in individual astronauts bone, muscle, etc.). It is noteworthy that the International Commis- While avoidance of the radi- sion of Radiation Protection (ICRP) has recently issued a report that ation risk is the best protective strategy, it is nearly impossible has important implications for two of the degenerative tissue risks, to avoid the radiation risk completely for astronauts. Therefore, circulatory diseases and cataracts In this re- countermeasures against adverse biological effects of space radi- cent review of early and late effects of radiation in normal tissues ation are necessary for the success of long term space missions.
and organs, it was concluded that for reactions manifesting very National Aeronautics and Space Administration (NASA) is primar- late after low total doses, particularly for cataracts and circulatory ily concerned with the health risks for astronaut exposures to GCR disease, it appears that the rate of dose delivery does not mod- and SPE radiation. SPEs occur with variable tissue dose-rates and ify the incidence, and for these two tissues, a threshold dose of doses, which range from 0 to 0.5 Gy/hour and 0 to 2 Gy, respec- 0.5 Gy was proposed For a NASA mission to tively, and with skin doses > 5 Gy NASA has Mars, fatal cancer risk has been considered the dominant risk in determined that the likelihood of acute risks during internal vehi- the past (considering the dose from GCR), but circulatory diseases cle activity is extremely small; however, there are scenarios during are likely to be of great importance in the newer risk estimates lunar, trans-lunar or Mars EVAs in which ARS may occur.
for a mission to Mars There have been a Acute radiation sickness has a sequence of a phased syndrome number of recent reviews or updates on the status of space radi- that varies with radiation dose, dose rate, quality and individual ra- ation research in the research areas of particular concern to NASA diation sensitivity which can for the exploration class missions planned for the future.
include nausea, vomiting, diarrhea and fatigue. These effects are For long term space exploration, bone loss and muscle atrophy manifested at approximately 4 to 24 hours post-exposure for expo- due to disuse are other major concerns related to the health of sures at sub-lethal doses, with a latency time inversely correlated the crew In ground- with dose. Since exposure to proton radiation, which represents based studies, disuse bone loss has been observed in the hindlimb the major type of radiation in an SPE, is known to induce ab- suspension rodent model normalities in leukocytes, erythrocytes and platelets there is also a reasonable concern for compromised with γ -rays exacerbates skeletal microarchitectural changes that immune functions, especially in the microgravity environment in are normally found during progressive, postpubertal aging prior to the onset of age-related osteoporosis Radi- Space ﬂight is known to alter immune ation exposure may increase the number of osteoclasts and the responses, and the causal factors include the stress due to in- extent of acute bone loss via increased reactive oxygen species creased radiation exposure production and oxidative damage, which implies different molec- and microgravity and non-load bearing sta- ular mechanisms from the bone loss caused by disuse Irradiation with 250 MeV protons followed by hindlimb The consistent effects on the immune suspension resulted in an approximately 20% loss of the trabec- system observed so far during space travel are as follows: reduc- ular bone volume fraction in the tibia and femur of irradiated tion in peripheral T-cell counts and a decrease in Natural Killer mice, and the mice receiving the combined treatment with proton (NK) cell number and functionality radiation and hindlimb suspension generally experienced greater decreases in cell-mediated immunity loss of the trabecular bone volume fraction, connectivity density, with altered cytokine production and trabecular number than either hindlimb suspension or irra- but normal levels of serum immunoglobulins diation treatment alone Irradiation with 56Fe An increased susceptibility to infection under ions, which represents a signiﬁcant component of GCR space ﬂight conditions has also been observed stimulates osteoclast differentiation even in the absence of The main concern of an impaired immune osteoblasts, thereby enhancing the sensitivity of bone cells to the system in the closed environment of a spacecraft is the altered effects of radiation Iron ion radiation con- ability to control bacterial, fungal, viral, and parasitic invasions tributes to a reduction in compressive strength and partially pre- vents the recovery of cancellous microarchitecture from adaptive and the loss of immuno- responses of lumbar vertebrae to skeletal unloading in hindlimb surveillance leading to tumor growth Counter- suspended mice Thus, irradiation with heavy measures that have been considered and/or evaluated for mitigat- ions may accelerate or worsen the loss of skeletal integrity trig- ing acute radiation effects on immune system include interferons, gered by musculoskeletal disuse in the microgravity environment.
which have a profound effect on the immune response both in There are some publications indicating that countermeasures may vivo and in vitro an active hex- be helpful to mitigate radiation induced adverse bone effects; for ose correlated compound, which activates immune function example, α-lipoic acid protects cancellous tissue from the detri- and enhances resistance to infection mental effects of irradiation and vitamin and mineral dietary supplementation, as The space radiation risks to the central nerve system (CNS) recently reviewed have been considered to be extremely important in the recent In addition to acute effects from radiation, there are numerous past due to major publications in this ﬁeld of research. Examples other major health concerns related to space radiation exposure.
of such publications include studies suggesting that the induction In the NASA Human Research Roadmap (A Risk Reduction Strat- of Alzheimer's disease may be a space radiation risk egy for Human Space Exploration), the Integrated Research Plan and attention deﬁcits may arise following exposure to (IRP) divides the space radiation risks into the following categories: low doses of space radiation Many other Risk of Acute and Late Central Nervous System Effects from Radi- publications have also indicated that there are major CNS space A.R. Kennedy / Life Sciences in Space Research 1 (2014) 10–43 radiation risks [e.g., ment of hepatocellular carcinoma There are some intriguing recent results in the radiation carcinogenesis ﬁeld Radiation exposure to of research. have indicated that there are distinct γ -rays or 56Fe ions signatures (transcriptome proﬁles) in normal human bronchial ep- ithelial cells exposed to γ -rays and different HZE particles. If this to have adverse effects on CNS and neurobehavior of irradiated an- effect can be conﬁrmed, it may give rise to studies in which the imals, including reduced performance in motor tasks and deﬁcits causative agent can be identiﬁed in human malignancies that could in spatial learning and memory. Alterations in neuronal function have been caused by radiation exposure. While the mechanism(s) in the HZE particle irradiated animals include reduced responsive- involved in space radiation induced carcinogenesis are still un- ness to agonist stimulation and increased Nigral cell loss, which known, there is evidence that space radiation induced oxidative parallel the neurobehavioral changes associated with aging stress is closely associated with carcinogenesis [e.g., The available data suggest that: a) the neu- It has been reported that space radiation induces per- rochemical and behavioral deﬁcits after HZE radiation exposure sistent oxidative stress in mouse intestine, which is likely to be have an apparent threshold below which there are no effects on associated with intestinal tumorigenesis There these endpoints, b) there does not appear to be a dose–response is evidence that space radiation induced carcinogenesis can be curve for many endpoints, such as upper body strength or radia- prevented or mitigated by several cancer chemopreventive agents, tion induced taste aversion learning, and c) there is no evidence which include antioxidants and protease inhibitors [as recently of spontaneous recovery of function that depends upon the in- reviewed retinoids [e.g. tegrity of the dopaminergic system after the HZE radiation ex- and fruit extracts posure It has been reported that persistent In addition, there are a number of new potential radiation induced oxidative stress is associated with space radi- cancer preventive agents that have been shown to mitigate in vitro ation induced CNS effects [e.g., In the CNS SEBs of the space radiation cancer risk; examples include mela- research area, there have been several publications indicating that tonin and a synthetic triterpenoid, bardoxolone countermeasures exist for some of the space radiation induced methyl, which protects against space radiation-induced transfor- adverse biological effects, which include melatonin or a metabo- mation of human colon epithelial cells lite lipoic acid There is extensive evidence that radiation exposure on earth fruit extracts, which ameliorate deﬁcits in behavior can give rise to cardiovascular diseases, as recently reviewed and signaling in rats irradiated with 56Fe ions and ﬂavonoid glycosides from Ginkgo biloba, myricetin and the research on heart and circulatory effects resulting from ex- quercetin (EGb761) which have been pos- posure to space radiation(s), it has recently been reported that tulated to improve cerebral metabolism, protect the brain against doses of 2 to 5 Gy 56Fe ion radiation targeted to speciﬁc arte- hypoxic damage and scavenge free-radicals rial sites in apolipoprotein E-deﬁcient (apoE−/−) mice accelerated Carcinogenesis has continued to be the major focus of the NASA the development of atherosclerosis In these stud- space radiation risk experimental investigations over the past sev- ies, it was concluded that 56Fe ions can promote the progression eral years, with most of the investigations not focused on the of atherosclerotic lesions to an advanced stage characterized by development of tumors in animals developing from space radia- compositional changes indicative of increased thrombogenicity and tion exposure(s), but instead focusing on various potential surro- instability. In numerous other studies, space radiation has been gate endpoint biomarkers (SEBs) of space radiation carcinogenesis.
shown to have detrimental effects on many other parameters re- There are some recent reviews that focus on the development lated to cardiovascular and circulatory diseases, with particularly of cancer in animals exposed to space radiation [e.g., strong effects leading to endothelial dysfunction [e.g., and angiogenesis [e.g., some recent individual re- ports on space radiation induced tumorigenesis [e.g., Risks of other degenerative diseases include radiation induced cataracts, as recently reviewed and some new hypotheses/thoughts concerning mechanisms of ra- diation carcinogenesis [e.g., It is noteworthy that the and risk estimates of radiation induced cancer [e.g., ICRP has recently proposed lowering the threshold for radiation induced cataracts to 0.5 Gy There have ing in the recent animal carcinogenesis studies is that 56Fe ions also been some recent studies on space radiation (or other sim- were not substantially more effective than γ -rays for the induc- ilar types of radiation such as heavy ion-cancer therapy [hadron tion of acute myeloid leukemia therapy]) induced cataracts It has been pointed out in numerous cur- rent and older reviews of space radiation carcinogenesis stud- has been reported that astronauts have an elevated risk of devel- ies that space radiation induced malignancies are dependent on oping cataracts which the species as well as the strain of the species used, and that has been associated with exposure to the high linear energy trans- a major task in this ﬁeld of research will involve determina- fer (LET) GCR present in the space environment.
tions about the appropriate methods to use for extrapolation of Current medical treatment for the acute radiation syndrome the space radiation induced cancer risks from experimental ani- routinely includes supportive care, antibiotics (quinolones and mal studies to humans. One example of the differences observed other agents), cytokine therapy, anti-emetic agents and analgesic in space radiation induced cancer studies concerns the develop- agents Other agents ment of hepatocellular carcinoma. While exposure to space ra- can also be used for the effects of the acute radiation syndrome, diation(s) has indicated a very high incidence of hepatocellular such as antihistamines, anti-inﬂammatories and radioprotectors carcinoma in one mouse strain Several FDA approved anti-emetic drugs, such in other experiments on space radiation induced as Kytril (granisetron), Zofran (Ondansetron), Decadron® (dexam- carcinogenesis using a different strain of mice, a dose of 0.5 Gy ethasone tablets) and Emend (Aprepitant) are known to prevent from 56Fe ions or 3 Gy from protons had no effects on the develop- or alleviate nausea and vomiting in patients or animals exposed A.R. Kennedy / Life Sciences in Space Research 1 (2014) 10–43 to radiation or chemotherapeutic agents of SPE radiation with conventional γ -ray radiation. The dose dis- tribution from electron radiation, however, can be manipulated to A systematic review and meta-analysis of 14 ran- simulate the dose distribution expected from SPE radiation. Mega- domized controlled trials, comprising 1451 patients, showed that voltage electron beam radiation has been utilized in the pig exper- amifostine (WR2721) signiﬁcantly reduced the side effects of ra- iments to accurately reproduce the total dose and dose distribution diation therapy In the animal studies, treat- of SPE protons The dosimetry involved in de- ment with amifostine was shown to protect against DNA damage termining simulated SPE radiation doses in pigs is illustrated in in cisplatin treated murine peripheral leukocytes which shows that the doses to the external organs (e.g., reduce changes in nucleolar morphology in- skin, lens) are very high, while the doses to internal organs (e.g., duced by cisplatin treatment and protect spinal cord, bone marrow) are quite low. The detailed methods for against cyclophosphamide-induced disruption of taste determining the organ doses are described elsewhere and methotrexate-induced small intestinal mucosi- These methods incorporate tis as well as inhibit tumorigenesis modern radiation oncology approaches of computed tomography Unfortunately, the severe side effects of am- (CT) based Monte Carlo dosimetry into the studies so that acute ifostine have limited its use in the space program for astro- biological effects in speciﬁc organ systems can be determined in nauts as well as in other human populations exposed to ra- animal model systems, and radiation toxicity from various types of diation. PrC-210 is a new aminothiol that has shown no de- SPE radiation exposures can be compared. This approach has also tectable nausea/vomiting or hypotension side effects in the fer- been used to predict the acute biological effects of SPE radiation ret and rat models in contrast to the strong exposure in astronauts. Ten full body human CT scans in various side effects of the current aminothiol, amifostine geometries have been analyzed to determine the impact of physical this compound shows promise as a new and environmental factors on organ dosimetry in humans. It has been found that, depending on the organ system of interest (deep In the past several years, we have been engaged in research vs. superﬁcial) and the ﬂuence/energy proﬁle of the exposure (hard to assess whether there will be adverse acute biological effects vs. soft event), either the physical size of the astronaut or the ﬂu- similar to those of ARS after exposure to the types of radiation ence/energy proﬁle for the SPE can be the determining factor for at the energies, doses and dose-rates expected during an SPE.
radiation dose/toxicity (Cengel, K.A., Schaettler, M.O., and Diffend- The overall objectives of our studies were to assess the risk of erfer, E., Unpublished data). In contrast, most of the experiments ARS and evaluate countermeasures for ARS, which can develop with mice or ferrets utilizing SPE-like proton radiation involved after exposure to SPE radiation. There is also a reasonable con- homogeneous proton radiation exposures with relatively low en- cern for a compromised immune system, due to high skin doses ergies like those present in SPEs; RBE values were then calculated from an SPE, which can lead to burns. Existing evidence sug- by comparison of the SPE-like proton results with those obtained gests that the best animal model for radiation induced vomit- in similar experiments using γ -ray radiation.
ing is the ferret whereas the In this review paper, the results of our studies on the bio- best animal model for radiation induced skin changes is the pig logical effects of several different types of space radiation, which include different types of SPE radiation, are discussed. Both acute Mice, on the other hand, are the most and chronic effects of space radiation have been evaluated in these frequently utilized mammalian species for evaluation of many ra- diation induced biologic effects. A major problem concerning theuse of mice for studies of SPE radiation is that mice do not 2. Acute radiation effects
vomit in response to irradiation There-fore, three species of animals, i.e., ferrets, pigs and mice, were The acute radiation effects evaluated in our studies include used in our studies to allow interspecies comparisons of the re- effects on hematopoietic cells, immune system effects (which in- sults obtained in the studies in several different areas of research, clude immune system changes resulting from a high dose of SPE whereas the effects on vomiting and skin were evaluated only in radiation to the skin), behavior/fatigue, heart functions, skin ef- the most appropriate animal species. Since astronauts will be ex- fects and organism survival after exposure to a lethal or potentially posed to space radiation in a microgravity environment, which is lethal dose of radiation.
known to cause bone loss, muscle atrophy and injury to soft con-nective tissues in animal models some of the radiation experiments with 2.1. Changes in hematopoietic cell counts after proton or conventional mice have been performed with and without partial weight sus- reference radiation exposures pension (PWS) at one-sixth G, which is known to be the gravityon the surface of the Moon or hindlimb sus- The changes in peripheral leukocytes in animals post-irradiation pension (HS), a model appropriate for mimicking travel in deep have been evaluated in ICR mice irradiated with 225-kVp X-rays space to evaluate and quantify the γ -rays from 60Co or possible synergy between radiation and simulated hypogravity on hematopoietic effects associated with ARS.
protons with energies of 50-MeV In the studies in which SPE radiation effects have been evalu- ated, it was considered extremely important to have comparable dose distributions between the SPE radiation and a standard ref- 78.4 MeV and 1-GeV pro- erence radiation in the animal model systems so that relative bio- tons as well as pro- logical effectiveness (RBE) values could be calculated tons with eight different energies between 31 and 75 MeV and SPE radiation is known to result in an inhomogeneous total simulated SPE protons with energies between 30 and 150 MeV body distribution, with a considerably higher dose delivered to the The changes in peripheral leukocytes have skin and underlying tissues than to the internal organs also been examined in ferrets irradiated with 60Co or 137Cs These characteristics of an SPE, which result in an unusual γ -rays and 110-MeV protons dose distribution pattern, make it diﬃcult to compare the results and Yucatan minipigs irradiated with 6-MeV electrons

A.R. Kennedy / Life Sciences in Space Research 1 (2014) 10–43 Fig. 1. Simulated dosimetry for Yucatan minipigs. Modern radiation oncology approaches, using CT based Monte Carlo dosimetry, have been incorporated into recent studies
to accurately predict speciﬁc organ doses from SPE radiation exposure in animals. Pig data are shown in the ﬁgures. A: 3D reconstruction from a pig CT image with the skin
rendered translucent to allow viewing of the internal organs (i.e., spinal cord [white], bone marrow [red], lung pleura [blue]). The organs were identiﬁed on separate CT axial
cross sections generating a 3D organ volume from the combined axial contours. (This ﬁgure has been reproduced with permission from Radiation Research B: Dose overlay on the 3D rendering of the whole body CT scan. (This ﬁgure has been reproduced with permission from Radiation Research C:
Dose overlay on a pig CT image (coronal plane cross-section) for 6 + 12 MeV electron irradiation (often called a Heat map or a Dose-Color map) (Doses were simulated for
6 + 12 MeV electron irradiation of a pig using a Monte Carlo based simulation algorithm (Varian Medical Systems, Palo Alto, CA). D: Dose–volume histogram (DVH ﬁgure)
illustrates organ radiation doses determined using the organ volumes and simulated dose distribution for the pigs receiving a skin dose of 20 Gy (from electrons). Note the
high skin dose in the dose–volume histogram (DVH) ﬁgure and the fact that the lens dose is also very high. (Images C and D – courtesy of Dr. Eric Diffenderfer.)
6 + 12-MeV electrons and SPE protons were still 64% and 76% below the respective control values whereas peripheral WBC and lymphocyte counts inthe mice irradiated with 5.9 Gy of 1-GeV protons were still ap- 2.1.1. Changes in hematopoietic cell counts in mice after irradiation proximately 37% and 44% below the respective control values in In an early study focused on determining the changes in circu- non-irradiated control animals In contrast, lating hematopoietic cell counts in mice exposed to SPE-like proton the PMN/neutrophil count was fully recovered by 8 weeks post- or γ -ray radiation (used as the reference radiation), ICR outbred irradiation with 8 Gy of 225 kVp X-rays mice aged 5–6 weeks were exposed to 60Co γ -rays at doses of In a separate study performed with male ICR mice aged 4–5 0.13, 0.25, 0.5, 1 or 2 Gy or spread out Bragg peak (SOBP) protons weeks, exposure to 1 GeV proton radiation administered in a sin- (50 or 70 MeV) at doses of 0.25, 0.5, 1 and 2 Gy. The radiation gle dose at low (5 cGy/minute) or high (50 cGy/minute) dose rates, exposures were delivered in a single dose at the low dose rate or in ﬁve fractionated doses at the low dose rate resulted in signif- of 0.5 Gy/hour or the high dose rate of 0.5 Gy/minute. The re- icant and dose dependent decreases in peripheral WBC and lym- sults demonstrated a dose dependent decrease in white blood cell phocyte counts at 24 hours post-irradiation (WBC) counts in mice exposed to high and low dose rate proton However, the difference among animals irradiated with the ﬁve and γ -radiation fractionated doses, or in a single dose at low or high dose rate, did In 4–5 weeks old ICR mice, peripheral WBC, lymphocyte and/or not reach statistical signiﬁcance at any of the doses evaluated, and polymorphonuclear leukocyte (PMN) counts decreased signiﬁcantly neither the WBC counts nor the lymphocyte counts in the animals at 4 and 24 hours after total body irradiation with 1 or 8 Gy of irradiated with 2 Gy 1-GeV protons in the ﬁve fractionated doses, 225 kVp X-rays or 1 or 5.9, 6.8 or 7.2 Gy of or in a single dose at low or high dose rate, were signiﬁcantly 1 GeV protons At 24 hours after irradiation different from the animals irradiated with 2 Gy of 51.24-MeV pro- with 8 Gy of 225 kVp X-rays, the neutrophil count was decreased tons administered at a low dose rate (5–7 cGy/minute), although to an average of 450 cells/μl; neutrophil counts of <500 cells/μl are they were all signiﬁcantly below the control WBC and lymphocyte clinically signiﬁcant. At 8 weeks post-irradiation, peripheral WBC counts in sham irradiated animals. These results suggest that the and lymphocyte counts in the mice irradiated with 8 Gy of X-rays effect of proton irradiation on the WBC and lymphocyte counts in A.R. Kennedy / Life Sciences in Space Research 1 (2014) 10–43 the irradiated mice is not altered by dose fractionation, dose rates evaluate the impact of hypogravity on the effect of SPE radia- or proton energy in the ranges evaluated tion on immunological function, experiments were performed with Some experiments were performed to determine whether age 6–8 weeks old female ICR mice that were irradiated with 0.5, 1 or sex/gender differences affected the ability of SPE-like radiation or 2 Gy of γ -rays with or without hypogravity simulated using to affect circulating hematopoietic cell counts in mice. To deter- the PWS model, described by The combi- mine the effects of age on this endpoint, 1-year old ICR mice nation treatment with PWS and γ -ray irradiation decreased total were exposed to proton or γ -ray radiation at doses of 0.0, 0.5, splenic lymphocyte viability in a dose dependent manner, and the 1.0, or 2.0 Gy, with a dose rate of 0.5 Gy/min suppressed splenic lymphocyte viability in groups exposed to a Whole blood samples were collected 2 Gy dose of radiation persisted for 4 days, which was the last up to 30 days post-irradiation and complete blood counts were an- time point evaluated in the study In addi- alyzed using automated technology as previously described tion, the viability of splenic lymphocytes was signiﬁcantly lower in the mice that received a 1 Gy dose of γ -rays in combina- A comparable experiment was performed in young tion with PWS treatment than in the mice that received a 1 Gy ICR mice, aged 6–8 weeks. Statistical analyses and RBE values were dose of γ -rays without PWS treatment on Day-1 or Day 4 post- determined as previously described irradiation. Treatment with PWS alone did not signiﬁcantly af- for the experiments performed in both young and aged mice. The fect the splenic lymphocyte viability at any of the time points older mice in this experiment were speciﬁcally aged to simulate evaluated up to 4 days. These results suggest that simulated hy- healthy, non-smoking, middle-aged astronauts. For example, the pogravity might have made splenic lymphocytes more sensitive preferred age range for European Space Agency applicants is 27–37 to the cell killing effects of radiation. In addition, results from years old. The decline in WBCs, neutrophils, lymphocytes and gran- these studies indicated that T cell activation was decreased in the ulocytes were not different using aged mice as compared to the irradiated mice (1 or 2 Gy) with or without simulated hypograv- reduction observed in young mice (Sanzari, J.K. and Kennedy, A.R., ity (PWS). Similar results were observed in experiments using Unpublished data). The lymphocyte nadir was at 2 days post- mice exposed to SPE-like proton radiation with and without sim- proton irradiation, with counts as low as 50%, 60%, and 77% of ulated hypogravity produced by HS the control values after exposure to 0.5, 1.0 and 2.0 Gy protons, re- In these experiments, mice were suspended spectively. The decline in lymphocyte counts after γ -ray radiation prior to and after SPE proton radiation exposure and total leuko- exposure was similar to that observed after the proton radiation cyte numbers and splenic lymphocyte functions were evaluated exposure, which were also comparable to the decline observed in on days 4 or 21 after the radiation exposure with and without young mice. The results for the granulocyte counts were also con- HS. Splenic lymphocyte subpopulations were altered at both time sistent between the aged and young mice, with a bimodal decline points investigated. At 21 days post-exposure, T cell activation observed for the ﬁrst nadir at 4 days post-irradiation (γ -ray or and proliferation were assessed in isolated lymphocytes. In these proton) and the second nadir at 16 days post-irradiation. The RBE studies, T cell activation was suppressed in the proton-irradiated values at different time points and for each dose were calculated animals and in the irradiated animals exposed to HS. From both in the aged and young mice. It was observed that the RBE val- types of experiments described above, the results suggest that ues were not signiﬁcantly different from 1.0 in either the young these irradiated animals with or without additional exposure to or the aged mice. In similar experiments, the radiation effects on simulated microgravity would have immune system suppression WBCs, lymphocytes and granulocytes were shown to be similar be- resulting from the lack of T cell activation. However, the peripheral tween male and female CHH/HeN mice blood cell (lymphocyte and granulocyte) counts were signiﬁcantly The only sex/gender difference observed in these experiments was higher in proton irradiated mice with HU treatment than without that non-irradiated male mice had 13% higher platelet counts and HU treatment and the HU treatment did not signiﬁcantly interact more enhanced recovery of platelets on day 16 after irradiation as with the proton radiation dose in the blood cell count data, indi- compared to female mice. Thus, it is conceivable that this differ- cating that the effects of radiation and hypogravity on peripheral ence between male and female mice could inﬂuence the response leukocytes were simply additive (or subtractive) with no signiﬁcant of platelets to total body radiation exposure.
In a study conducted with 5 to 7-weeks old female ICR mice, To determine the RBE values for the effects of SPE protons in exposure to protons with 8 energy levels ranging from 30.63 to hematopoietic cells, several studies were performed in mice us- 74.52 MeV or simulated SPE protons with energies ranging from ing 21-MeV electrons 60Co γ -rays 30 to 150 MeV at high (0.5 Gy/minute) or low (0.5 Gy/hour) dose or 137Cs γ -rays rate resulted in signiﬁcant decreases in peripheral WBC and lym- as the reference radiations. In male ICR mice irradiated with 2 phocyte counts as early as 4 hours post-irradiation Gy of 70-MeV protons or 21-MeV electrons, peripheral WBC, lym- At 24 hours post-irradiation with the 30.63–74.52 MeV pro- phocyte and granulocyte counts decreased signiﬁcantly in the ir- tons, the dose response curves were nearly identical between the radiated mice as compared to the sham irradiated controls, but mice irradiated at the low and high dose rates for WBCs or lym- the differences between the proton and electron irradiated mice phocytes. In a separate experiment performed in the same series were not statistically signiﬁcant for WBCs, lymphocytes or granu- of experiments, the mice were irradiated with protons at a very locytes In 5 to 6-week old female ICR mice low dose rate of 0.28 cGy/minute (17 cGy/hour), and the results irradiated with 60Co γ -rays, 50-MeV protons or 70-MeV protons were similar to those of the mice irradiated at the high dose rate at low (0.5 Gy/hour) or high (0.5 Gy/minute) dose rate, the pe- These results again indicate that dose rate ripheral WBC count decreased in a dose-dependent manner at 24 in the range evaluated has little or no impact on the suppressive hours post-irradiation and the RBE values for 50-MeV and 70-MeV effects of proton radiation on the peripheral WBC or lymphocyte protons at either low or high dose rate were not signiﬁcantly counts in irradiated mice.
different from 1 with 60Co γ -rays as the reference radiation at During space missions, astronauts are potentially exposed to the corresponding dose rates In a separate SPE radiation in a reduced gravity environment. Thus, several ex- study performed with 6-week old female ICR mice, the neutrophil periments have been performed to determine the effects of sim- count was monitored for 30 days post-irradiation with 0.5, 1 or ulated microgravity on blood cell counts with or without addi- 2 Gy of 137Cs γ -rays or 74-MeV protons at dose rates of 0.44 tional exposure to space radiation [e.g., To Gy/minute and 0.5 Gy/minute, respectively A.R. Kennedy / Life Sciences in Space Research 1 (2014) 10–43 signiﬁcantly at 48 hours post-irradiation (p < 0.001), both in adose-dependent manner.
In addition to the radiation dose, the dose rate also affected the WBC and neutrophil counts at 3 hours, but not at 48 hours post-irradiation, when the WBC and neutrophil counts in the groupsirradiated at HDR were lower by approximately 16% and 32%, re-spectively, as compared to the WBC and neutrophil counts in thegroups irradiated at the LDR The dose rateeffects for the WBC and neutrophil counts were small, as com-pared to the magnitude of the effect of radiation dose at 3 hourspost-irradiation, and disappeared by 48 hours post-irradiation, sug-gesting that the higher dose rate might have only accelerated theonset of the radiation effects, but did not affect the overall mag-nitude of the radiation effects that developed at the later timepoints. Given the fact that the dose rate effect was not observedat 48 hours post-irradiation, when more pronounced losses of cir-culating WBCs, neutrophils and lymphocytes were observed in the Fig. 2. Changes in neutrophil counts of mice exposed to a 2 Gy dose of SPE proton or
irradiated ferrets, the dose rate effect probably did not have a γ -ray radiation. As shown in the ﬁgure, the effects of SPE like proton radiation on biologically meaningful impact on the blood cell counts in the ir- circulating neutrophil counts of mice are approximately the same as those of γ -ray radiated animals.
radiation. In this ﬁgure, the absolute neutrophil counts are given at various times RBE values were determined for 110 MeV protons using 60Co post-irradiation. In radiation therapy patients, when the white blood cell countsfall below the level of 500 cells/microliter, it would trigger a medical response and γ -rays as a reference radiation and peripheral leukocyte counts in the patients would be considered as candidates for countermeasures (e.g., Neulasta the irradiated ferrets as the biological endpoints. The RBE values treatment). Thus, after irradiation of the mice with either SPE-like proton or γ -ray derived from the WBC counts for 110 MeV protons delivered at the radiation, the neutrophil counts fall to critically low values (< 500 cells per mi- high or low dose rates at 3 and 48 hours post-irradiation ranged croliter). (Courtesy of Dr. Ana Romero-Weaver; data from from 1.19 to 4.02 at 0.75 Gy and declined with the increase in theradiation dose to a narrow range of 0.59 to 1.04 at 2 Gy For the 2 Gy γ -ray and proton dose groups, the neutrophil The RBE values calculated from the lymphocyte count counts decreased by approximately 65% and 70%, respectively, at data for the 110 MeV protons at the same time points and dose the ﬁrst nadir, which occurred on Day-4 post-irradiation, and then rates were within a range of 0.83 to 1.41 at 0.75 Gy and showed a fully recovered by Day-10 post-irradiation to levels that were not slightly downward trend with the increase in the proton radiation signiﬁcantly different from the pre-irradiation control level. The dose to a narrow range of 0.67 to 0.84 at 2 Gy. With only a few neutrophil counts for the 2 Gy γ -ray and proton dose groups de- exceptions, a similar downward trend was also observed for the creased again by approximately 80% and 75%, respectively, at the RBEs based on the neutrophil, monocyte and eosinophil counts in second nadir, which occurred by Day-16 post irradiation, and then the irradiated ferrets. These results suggest that 110-MeV protons fully recovered again by Day 23 post-irradiation. Comparable re- might be more effective than 60Co γ -rays in reducing the periph- sults were observed in similar studies utilizing 74-MeV protons eral leukocyte counts at the low end of the radiation dose range and 137Cs γ -rays in which the changes in WBC counts were evalu- evaluated in ferrets.
While the early radiation induced changes in the blood cells ated over time The results from the of ferrets were similar to those in the irradiated mice, the later studies in mice indicate that proton radiation is not more effective portion of the time course was quite different between mice and than the commonly used reference radiations with respect to ef- ferrets. In the irradiated mice, the initial sharp decrease in WBC fects on hematopoietic cells counts post-irradiation is followed by a gradual recovery to base- Overall, the results show that the RBE values are not signiﬁ- line levels by 30 days post-irradiation. However, the recovery in cantly different from 1 when the effects of SPE-like protons are the WBC counts did not occur in ferrets exposed to radiation at compared to those from the reference radiations (γ -ray or electron doses of 1.5–2 Gy; 2 Gy was the highest dose of proton or gamma radiation) in mice, and that SPE-like protons and γ -ray radiation radiation evaluated in ferrets. This was shown to be due to the de- result in almost identical dose–response curves over time, as illus- velopment of disseminated intravascular coagulation trated in [data in this ﬁgure are from as discussed below.
2.1.3. Changes in peripheral leukocyte counts in Yucatan minipigs after 2.1.2. Changes in peripheral leukocyte counts in ferrets after irradiation In ferrets irradiated with up to 2 Gy of 60Co γ -rays or 110-MeV In the Yucatan minipigs irradiated with 6-MeV electrons at a protons at a high dose rate (HDR) of 0.5 Gy/minute or a low dose total skin dose of 25 Gy, the WBC count decreased signiﬁcantly rate (LDR) of 0.5 Gy/hour, the white blood cell (WBC) count de- on day-1 post-irradiation, and then recovered by day-7 and in- creased signiﬁcantly within 3 hours after the radiation exposure, creased signiﬁcantly above the pre-irradiation level by day-30 after and the average magnitude of the WBC decrease in the groups irradiation The WBC count did not change irradiated with 2 Gy of γ -ray or proton radiation at 48 hours post- signiﬁcantly in any other dose groups irradiated with electrons at irradiation was approximately three times the decrease observed a dose of 15 Gy or below. The lymphocyte count in the minip- at 3 hours post-irradiation The lymphocyte igs decreased signiﬁcantly as early as 4 hours post-irradiation with count also decreased signiﬁcantly within 3 hours after irradiation 6-MeV electrons at skin doses of 15 or 25 Gy and then recov- with 60Co γ -rays or 110-MeV protons, but the magnitude of the ered to the pre-irradiation level by day-14 and day-7 for the 15-Gy decrease was similar at the two time points (3 and 48 hours) and 25-Gy dose groups, respectively. The neutrophil count did not post-irradiation. In contrast, the neutrophil count increased signif- change signiﬁcantly after exposure to the electron irradiation in icantly at 3 hours post-irradiation (p < 0.001) and then decreased any dose groups at any time points except for a two-fold increase A.R. Kennedy / Life Sciences in Space Research 1 (2014) 10–43 in the neutrophil count in the 25-Gy dose group at day-30 afterirradiation.
In the Yucatan minipigs exposed to radiation with 6 + 12 MeV electrons, which is a suitable reference radiation with compara-ble body dose distribution as the SPE radiation the WBC count decreased signiﬁcantly within a day post-irradiation in the 10, 15 and 20 Gy dose groups, but not in thegroups irradiated at a dose of 7.7 Gy or below Between day-1 and day-7 post-irradiation, the WBC countreached the lowest level, and then recovered slowly thereafter. Byday-30, the WBC counts in the 10, 15 and 20 Gy dose groups allrecovered to levels that were not signiﬁcantly different from thebaseline value. The signiﬁcant decrease in lymphocyte counts oc-curred earlier, to a greater extent and extended to lower dose (i.e.,5, 7.5 and 7.7 Gy) groups than the decrease in the WBC count afterthe 6 + 12 MeV electron irradiation. Within a day after irradiationwith 6 + 12 MeV electrons at doses up to 20 Gy, the lymphocytecount decreased by up to 77%. By day-30 post-irradiation, the lym-phocyte count in the 5, 7.5, 7.7 and 15 Gy dose groups recovered Fig. 3. Changes in WBC counts of pigs exposed to eSPE or pSPE radiation with a skin
dose of 7.7 Gy. The pSPE dose involved several different energies of SPE radiation, as
to levels that were not signiﬁcantly different from the baseline described previously and the electron exposure involved two value; however, the lymphocyte counts in the 10 and 20 Gy dose different energies of electrons (6 and 12 MeV electrons to result in a dose distribu- groups were still signiﬁcantly below the baseline level. The neu- tion like that expected for the SPE radiation exposure). The last time point for the trophil count in the Yucatan minipigs irradiated with 6 + 12 MeV evaluation of the changes in the circulating white blood cell numbers was 30 dayspost-irradiation. It can be observed in the ﬁgure that pig white blood cells return to electrons did not show a consistent pattern of change among dif- normal levels by 30 days post-exposure to electron radiation, but do not return to ferent dose groups post-irradiation. Both increases and decreases normal levels in proton irradiated pigs over the same time period. Signiﬁcant dif- in the neutrophil counts were observed at different time points ference as determined by one-way ANOVA followed by the Tukey Test is indicated post-irradiation with 6 + 12 MeV electrons at skin doses up to 15 by ∗ (p < 0.05), ∗∗ (p < 0.01) or ∗ ∗ ∗ (p < 0.001).
Gy, although the changes did not reach statistical signiﬁcance dueto the relatively large variations in the control group and in some to proton simulated SPE radiation do not return to normal levels of the irradiated groups.
over this time period.
In the Yucatan minipigs irradiated with SPE-like protons with Based on the WBC, lymphocyte and neutrophil count data for energies of 155 MeV or below, the WBC count decreased signiﬁ- the minipigs exposed to 6 + 12 MeV electron radiation cantly within a day post-irradiation with a single skin dose of up and simulated proton SPE radiation to 10 Gy Between day-1 and day-4 post- RBE values were calculated for the effect of simulated pro- irradiation, the WBC count reached the lowest level, and then ton SPE radiation on leukocytes in irradiated animals. The results recovered slowly thereafter. By day-30, the WBC count was no show that the RBE value for the simulated SPE radiation varied more than 38.3% below the baseline level for the animals irradi- with both the radiation dose and the time post-irradiation ated with 5, 7.7 or 10 Gy doses of protons. By day-90, the WBC For WBC counts, the RBE calculated for the simu- count recovered fully for the 5 Gy radiation dose group while re- lated SPE radiation displayed a downward trend with the increase maining 18.7% and 33.5% below the baseline level for the 7.7 and in radiation dose on Day-1, Day-4 and Day-14 post-irradiation. At 10 Gy radiation dose groups. Signiﬁcant decreases in lymphocyte the 5 Gy proton dose level, the RBE values for the simulated SPE counts occurred earlier and to a greater extent than the decrease radiation were 2.0, 4.1 and 3.3 on Day-1, Day-4 and Day-14, re- in the WBC count for the minipigs irradiated with 5, 7.7 and 10 spectively, after irradiation For lymphocyte Gy of protons. On day-1 after irradiation, the lymphocyte count counts at 4 hours post-irradiation, the RBE for the simulated SPE reached the lowest level, which was 73.0%, 79.7% and 89.5% below radiation also showed a downward trend with increasing dose, the baseline level for the 5, 7.7 and 10 Gy radiation dose groups, with the RBE value changing from 9.6 at 5 Gy to 4.6 at 10 Gy.
respectively. By day-30 post-irradiation, the lymphocyte count in However, the RBE trend for the simulated SPE radiation calculated the irradiated animals was not more than 34.5% below the base- from the lymphocyte count data was relatively ﬂat on Day-1 and line level. By day-90, the lymphocyte count recovered fully for the Day-4 and only slightly downward with the increase in the radia- 5 Gy radiation dose group and was not more than 19.5% below the tion dose on Day-14 post-irradiation. For neutrophils, the RBE for baseline level for the two higher radiation dose groups.
the simulated SPE radiation also displayed a noticeable downward The neutrophil count change in the Yucatan minipigs displayed trend with the increase in radiation dose on Day-4 and Day-14.
quite a different time course as compared to the changes ob- The ﬁtted RBE values were higher than 1.00 at all three simulated served in the WBC and lymphocyte counts. The neutrophil count SPE radiation dose levels of 5, 7.7 and 10 Gy for WBCs and lym- increased by up to 79.8% at 4 hours post-irradiation, and then de- phocytes, and the lower limits of the 95% conﬁdence interval for creased by up to 42.1% on day-1 after irradiation. The neutrophil the RBEs were above 1.00 for all dose levels on Day-1, 4 and 14 count reached the lowest level between day-4 and day-14 post- except for 10 Gy on Day-1 for WBCs and 7.7 and 10 Gy on Day-4 irradiation, and then recovered slowly thereafter. By day-90 post- for neutrophils. In addition, the ED10 and ED50 values for the sim- irradiation, the neutrophil counts in the 7.7 Gy and 10 Gy dose ulated SPE proton radiation were signiﬁcantly lower than those for groups were still 29.3% and 48.0% below the pre-irradiation level.
the 6 + 12 MeV electron radiation on Day-1, 4 and/or 14 post- The results for the pig WBC counts over a 30 day experimental pe- irradiation for WBCs, lymphocytes and/or neutrophils riod following exposure to proton or electron radiation are shown These results indicate that simulated SPE proton radia- in It should be noted that, following exposure to electron tion is signiﬁcantly more effective than 6 + 12 MeV electrons with simulated SPE radiation, the WBC counts return to normal levels respect to the effects on the peripheral WBCs, lymphocytes and by 30 days post-irradiation, but the WBC counts in pigs exposed neutrophils, especially at the low end of the radiation dose range A.R. Kennedy / Life Sciences in Space Research 1 (2014) 10–43 evaluated. It was observed that the neutrophils ware particularly radiation may be far more hazardous to humans than previously sensitive to the damaging effects of proton radiation.
estimated from studies performed with small animals.
2.1.4. Summary of effects of SPE radiation on hematopoietic blood cell 2.2. Immune system effects counts in ferrets, mice and pigs For hematopoietic cells, signiﬁcant decreases in white blood 2.2.1. Effect of SPE-like radiation on gastrointestinal tract integrity cell counts were observed in mice and ferrets irradiated at high Numerous studies have been performed to evaluate the effects (∼0.5 Gy/minute) and low (0.5 to 0.17 Gy/hour) dose rates, starting of SPE radiation on the immune system as a part of research on the at doses of 0.5 Gy and up to 2 Gy. In some, but not all, of the stud- acute risks of SPE radiation exposure ies involving dose-rate comparisons, lowering the dose rate had a small, but statistically signiﬁcant, sparing effect on the white blood the SPE radiation studies were performed with and without mi- cell count parameters evaluated, but in many other dose-rate ex- crogravity simulated with PWS or HS periments performed, such changes were not observed. Thus, over the range of dose-rates evaluated, it is concluded that lowering the For the studies related to immune sys-tem effects, mice were exposed to homogeneous doses of either radiation dose-rate does not produce sparing effects on hematopoi- γ -ray or SPE-like proton radiation. The effects of γ -ray and SPE- etic cell counts that are of biological signiﬁcance. At the higher like proton radiation were comparable in these studies, and none doses of proton or γ -ray radiation, the neutrophil counts in the of the observed effects described below were speciﬁc to proton or blood of both mice and ferrets reach critically low levels (< 500 γ -ray radiation.
cells per microliter) The gastrointestinal (GI) tract contains over 1012 bacteria, and If such a low value occurred in a patient in a hospital these bacteria have many important functions including carbohy- (e.g., following radiation or chemotherapy for cancer), this would drate fermentation and absorption, repression of pathogenic mi- trigger a medical response, and suggest the use of countermea- crobial growth, and continuous modulation of the gut and sys- sures to increase the level of neutrophils. For the pigs, there were temic immune system. A critical function of the GI tract is the also highly signiﬁcant reductions in the levels of WBCs following containment of commensal bacteria, which involves the control proton or electron SPE radiation exposure, but they did not reach of bacteria and bacterial product passage across the GI mucosa, the critically low values observed in the mice or ferrets at any of known as bacterial translocation; this function can be disturbed in the high skin doses evaluated.
many different diseases. In a study performed with ICR mice at A major difference in the pig response to the electron and pro- 5–6 weeks of age, irradiation with 2 Gy of 50 or 70-MeV protons ton SPE radiation was that the neutrophil count did not show a resulted in a transient increase of lipopolysaccharide (LPS) in the meaningful recovery by 3 months after exposure to 10 Gy of pro- serum at one day post-irradiation, and the increase was accompa- ton SPE radiation . For pigs exposed to 10 or nied by increases in acute-phase reactants, such as lipopolysaccha- 20 Gy of electron SPE radiation, the neutrophil count recovered as ride binding protein (LBP) and soluble CD14 (sCD14), circulatory expected within a month after the radiation exposure pro-inﬂammatory cytokines (including TNF-α, IL-1β and IL-6), and These results indicate that the pigs might be less ca- transient disruption of tight junctions in the GI track, which in- pable of repairing the DNA damage caused by the proton radiation dicated a transient increase in bacterial translocation across the exposure than the DNA damage caused by the electron radiation at GI tract and systemic activation of the innate immune system similar or higher doses. Similar results were observed for mice ex- HS was also shown to cause a breakdown in the posed to proton total body radiation at a dose of 5.5 Gy, which containment of Gram negative bacterial products, as measured by signiﬁcantly suppressed the neutrophil count at 9 weeks post- circulating LPS The combined treatment of a 2 Gy irradiation In contrast, the neutrophil count dose of either γ -ray or SPE-proton radiation with HS led to a in mice exposed to x-rays at comparable or considerably higher greater and more sustained elevation in the level of LPS in the doses (up to 8 Gy) was fully recovered at 8 weeks post-irradiation serum. Bacterial translocation is known to increase circulating lev- These results suggest that mouse neutrophils els of LPS and other bacterial components, which include bacterial might also be more sensitive to the DNA damaging effects of pro- DNA To determine whether the increase in ton radiation when given at a relatively high dose (5.5 Gy). At a LPS induced a systemic response, LBP, which is a type 1 acute dose of 2 Gy, the neutrophils in the irradiated mice appeared to phase protein, was measured in ICR mice exposed to radiation be equally sensitive to the damage from proton and γ -ray irradia- with and without hindlimb suspension. LBP is a circulating pro- tein that binds to LPS of Gram-negative bacteria; it is constitutively We have calculated RBE values for SPE-like radiations using present but can be induced to higher levels during various types hematopoietic cell count data from mice, ferrets and Yucatan of infection and inﬂammatory processes. LBP was increased after minipigs irradiated with SPE-like proton radiation and a suitable treatment with proton radiation or HS and was increased further reference radiation (e.g., γ -rays, x-rays or electrons). A higher RBE when these stressors were combined Similar value for a given biological endpoint, such as blood cell count, indi- results were observed for sCD14, another very sensitive marker of cates that the SPE-like proton radiation is more effective than the increased levels of circulating LPS. Circulating levels of interferon- reference radiation in affecting that biological endpoint. In these alpha (IFN-α) were measured, and at least additive levels of IFN-α studies, it was observed that there were: 1) different RBEs for dif- were observed for mice treated with both radiation (2 Gy of γ -ray ferent biological endpoints in the same animal species/strain, and or proton radiation) and HS. These results demonstrate that cir- 2) different RBEs for the same endpoint in different species/strains.
culating LPS, resulting from exposure to SPE-like radiation, HS or The RBE values estimated based on the WBC results vary greatly both, led to a systemic response. It has been concluded from these between mice, ferrets and pigs, with the RBE values being greater studies that there is a synergistic effect when hindlimb-suspended in ferrets than in mice at times up to 48 hours post-irradiation mice are additionally exposed to SPE-like radiation.
and considerably greater in pigs than in To determine the mechanisms involved in the increased bacte- ferrets and mice This trend suggests that rial translocation across the GI tract, immunohistochemical stain- the RBE values for WBC counts in humans could be considerably ing for the tight junction protein, Claudin-3, was performed on greater than those observed in smaller mammals, and SPE proton terminal ileum sections of mice, and a signiﬁcant increase in the

A.R. Kennedy / Life Sciences in Space Research 1 (2014) 10–43 Fig. 4. Proton radiation induces breaks in the GI epithelial barrier. Terminal ileum obtained 2 days post-irradiation from a control mouse (A) or a mouse irradiated with
2 Gy of protons at the low dose rate (B), and stained for Claudin-3. Black arrows show regions of tight junction incongruity. Original magniﬁcation 400×. Reprinted with
permission from Radiation Research
Fig. 5. SPE-like proton radiation and hindlimb suspension lead to the accumulation of LPS in subepithelial regions of the ileum. Terminal ileum obtained 4 days post irradiation
and/or 6 days post hindlimb suspension or from control animals was stained for LPS using a mouse mAb speciﬁc for E. coli LPS. A: represents ileum from a control mouse,
B: ileum for a mouse irradiated with 2 Gy of 70 MeV protons, C: ileum from a mouse subjected to hindlimb suspension, and D: ileum from a mouse subjected to HS and
irradiated with 2 Gy of 70 MeV protons. It can be observed in the ﬁgure that the amount of LPS accumulated in the subepithelial region of the ileum is considerably greater
in the mouse exposed to both HS and SPE proton irradiation than in mice exposed to either HS or SPE proton radiation alone. Original magniﬁcations – 200×.
A.R. Kennedy / Life Sciences in Space Research 1 (2014) 10–43 number of breaks and reductions in staining were observed in erance", in which subsequent responses are reduced in quality and mice exposed to proton or γ -ray radiation and HS, as illustrated in quantity, which is thought to protect the host by limiting excessive These studies indicate that SPE-like radiation and hindlimb inﬂammation and preventing septic shock suspension induced breaks in the GI epithelial barrier, and sug- As has been observed in many different dis- gest that the increased frequency of breaks could be responsible mechanistically for the increase in translocation of bacterial prod- ucts. To further substantiate this association, the terminal ileum continuous or extended exposure to PAMPs was stained with two antibodies that recognize LPS, a mouse mon- can lead to long-lived immune dysfunction. This condition may ex- oclonal antibody against E. Coli (j5) LPS and a goat anti-lipid A ist during space ﬂight, as LBP, a well-known marker of immune ac- IgG that cross-reacts with Pseudomonas aeruginosa, Klebsiella pneu- tivation, is known to be elevated in astronauts' plasma moniae, E. coli 0157, Salmonella enteriditis, Enterobacter aerogenes, It is possible that the elevated blood E. Hermanii, Yersinia enterocolitica and Shigella sonnei. Control an- levels of LBP resulting from exposure to SPE radiation and simu- imals demonstrated no LPS speciﬁc staining in the intervillous lated microgravity (HS) may bring about immune dysfunctions of space, while low levels were observed in mice treated with SPE- the sort that have been observed during and after extended space- proton radiation or HS alone. For the mice exposed to the com- bined treatment with SPE-proton radiation and HS, there was ahigh level of diffuse staining, as shown in Thus, the conclu- 2.2.2. Effect of SPE-like radiation on skin immune function sion from these studies was that SPE-like radiation and hindlimb Two models were used to evaluate the effects of SPE radiation suspension induced the accumulation of LPS in subepithelial re- on skin immune function. One involved Yucatan minipigs, as their gions of the ileum.
skin histological structure is an accurate model of human skin, and It has been estimated that astronauts could receive a dose of up the other involved mice, in which in depth investigations of alter- to 2 Gy to the bone marrow from SPE radiation ations in immune function could be performed. A major difference As discussed above, when a 2 Gy dose of radi- between the models used involves the depth of radiation penetra- ation is combined with simulated microgravity, an enhanced and tion. The pigs were exposed to electrons or protons as an SPE-like prolonged impairment of commensal bacteria containment was inhomogeneous dose of radiation, with a relatively high dose de- observed. We have identiﬁed the mechanism for the loss of con- livered to the epidermis and dermis and lower doses delivered to tainment, which is that radiation plus HS leads to breaks in the internal organs while the mice received proton or tight junctions between GI tract epithelial cells, which results in reference radiation (γ -rays) exposures as a homogeneous dose of the migration of LPS into the subepithelial tissue. Potential ther- radiation. The pigs were exposed to skin doses as low as 2.5 Gy to apies to treat this immune defect could target the GI defect that up to 25 Gy, which was the highest skin dose evaluated in these leads to bacterial translocation or by reducing the inﬂammatory studies. It should be pointed out that skin doses for major histori- activity of translocated bacterial products. The mucosal integrity of cal SPEs were calculated to be as high as 32 Gy the GI tract is maintained by a population of CD4+ cells that pro- however, such a high skin dose would represent a worst case sce- duce IL-17 (Th17 cells). Their loss is known to be correlated with nario that would not be expected to occur frequently. Delayed type increased bacterial translocation Although hypersensitivity (DTH) responses to phytohemagglutinin (PHA) and there are no current therapies that can mitigate the loss of GI Th17 LPS were measured prior to radiation and at 7, 14 and 30 days cells, this is an area of research worthy of investigation. Antibiotics post-irradiation. The responses were symmetric and recorded as can be used to treat the increased bacterial translocation, and they the average distance across for induration, erythema and ulcera- are known to be capable of reducing serum LPS levels tion. Since a similar pattern was observed for all doses of radiation used, with signiﬁcant increases in the response after radiation, but Numerous immune system alterations have been associated with no dose dependency, we analyzed all radiation dose groups with space ﬂight in humans and in animals during ground-based together for the response to control (PBS), PHA and LPS treatments.
spaceﬂight models (e.g. HS), as has been reviewed [e.g., A signiﬁcant enhancement in the DTH response to PHA was ob- served at all post-irradiation time points evaluated. The responses The major effects of spaceﬂight on the immune system to LPS were not signiﬁcantly elevated at day 7, but they become have been well-characterized, and include changes in cytokine statistically signiﬁcant at 14 and 30 days post-irradiation production, leukocyte subset distribution and antibody production The appearance of ulceration after radiation expo- Examples of cytokines released sure was noted for both PHA and LPS treatments. It is assumed in response to stimulation include the following: an increase in that ulceration occurred as part of the enhanced immune response anti-inﬂammatory cytokines and a decrease in TNF-a in LPS stim- post-irradiation. If radiation was responsible for the ulceration, ul- ulated spleen cells reductions in interferon-γ ceration should have increased with increasing doses of radiation, and IL-2 following phorbol 12-myristate 13-acetate and ionomycin which was not the case. Mice were exposed to a homogeneous stimulation of peripheral blood cells of astronauts dose of radiation up to a 2 Gy dose. Mouse skin challenged with and reduced NK cell number and function intradermal PHA was measured and a similar increase in DTH re- Such alterations in im- activity was noted after exposure to 2 Gy of proton or γ -ray radi- mune function are similar to those brought about when there is ation and (Weissman, D., Unpublished data)].
increased immune activation produced by exposure to pathogen Skin is known to contain high frequencies of FoxP3 expressing associated molecular patterns (PAMPs), such as LPS regulatory CD4+ T cells (Tregs). In the skin from irradiated pigs and mice, immunohistochemical analysis demonstrated a loss of CD3+ and CD25+ cells. To determine whether loss of Tregs was re- ciﬁc examples of this are as follows: a reduction in proinﬂamma- sponsible for the enhanced DTH responses, RNA was isolated from tory cytokine production by myeloid cells murine skin prior to and at 2, 7 and 14 days post-irradiation for a reduction in antigen-speciﬁc T cell effector quantitative PCR measurements of CD3, FoxP3 and GAPDH mRNA.
cytokine responses and a reduc- The results demonstrate that there was a statistically signiﬁcant tion in circulating NK cells The changes in reduction in FoxP3 mRNA at all 3 time points following expo- responses brought about by exposure to PAMPs are known as "tol- sure to 2 Gy of irradiation. Smaller decreases in CD3+ cells were

A.R. Kennedy / Life Sciences in Space Research 1 (2014) 10–43 observed, demonstrating that FoxP3 positive cells were being se-lectively lost (Zhou, Y., Ni, H., Balint, K., Sanzari, J.K., Dentchev, E.,Diffenderfer, E., Wilson, J., Kennedy, A.R., Cengel, K.A. and Weissman, D.,Unpublished data). Mouse skin was obtained at various time pointspost-irradiation in the experiment and single cell suspensions oflymphoid cells were obtained by enzymatic digestion for analysisof CD4, CD25 and FoxP3 cells by ﬂow cytometry. Statistically signif-icant decreases in the percent of CD4+ T cells expressing CD25 andFoxP3 were observed at all post-irradiation time points evaluated.
The greatest loss was observed at 4 days post-irradiation, with aslow increase in Tregs over the next 28 days. The proliferation ofskin CD4+ T cells increased with the loss of Tregs, demonstratinga functional effect.
The loss of skin Tregs could be due to the cell killing effects of radiation or alterations in traﬃcking. We examined splenic lym-phoid cells and observed that, with the loss of skin Tregs post-irradiation, there was a statistically signiﬁcant increase in the per-cent of Tregs in the spleen. The increase in Tregs led to a drop inthe proliferation of activated CD4+ and CD8+ cells. A recent reporthas indicated that Tregs traﬃc through skin and that they are themain types of cells exiting skin during inﬂammation In these studies, it was observed that half of the skin cellsthat migrated to draining lymph nodes were Tregs at steady state.
Tomura et al. also noted that when an immune reaction was in-duced in the skin, the frequency of Tregs draining to lymph nodesincreased signiﬁcantly and made up the majority of cells exitingthe skin In addition, it was found that theincrease in Tregs leaving the skin resulted in more suppression ofT cell activation in the draining lymph nodes and spleen whereTregs accumulated.
To identify mechanisms for the radiation induced depletion of skin T cells, inﬂammatory genes in mouse skin obtained prior toand at various time points after mice were treated with a sin-gle radiation dose of 2 Gy were analyzed. It was observed that Fig. 6. Survival of mice challenged with bacteria after irradiation with or without
at 6 hours post-irradiation, multiple acute inﬂammatory markers, hindlimb suspension. C3H/HeN mice were treated with HS, 2 Gy of SPE-like proton CXCl chemokines, were upregulated. At 24 hours and continuing radiation, or both, and then the mice were challenged with a non-toxic dose ofPseudomonas aeruginosa (Panel A) or Klebsiella pneumoniae (Panel B) bacteria. There through day 14, chronic inﬂammatory markers, CCL chemokines, was a high level of mortality observed in all treatment groups except the control complement and IL-10, were induced. These data indicated that group; these mice were not exposed to HS or radiation, but were given the bacterial irradiation at a dose of 2 Gy induces long-lived inﬂammatory challenge. (The images were provided by Drew Weissman, M.D., Ph.D. [data from changes in the skin, including alterations in chemokines known to attract Tregs to the site of infection, including CCL17 and CCL22which are down-regulated. We hypothesize fection in an astronaut during the Apollo 13 mission that the radiation induced inﬂammation establishes an environ- ment that induces T cells, namely of the regulatory phenotype, to As the control of infections during spaceﬂight is leave the skin and reduces their ability to return to the skin. The a major problem, much effort has been focused on determining lack of Tregs in the skin likely results in a loss of control of the the effects of spaceﬂight stressors, such as simulated microgravity inﬂammatory response induced by PHA and LPS challenges, result- and SPE-like radiation, on the ability to defend against a bacte- ing in enhanced responses post-irradiation. The clinical signiﬁcance rial challenge In these studies, mice were ex- of this for an astronaut exposed to SPE radiation is unknown. Po- posed to SPE-like radiation and/or HS, and then challenged with tentially, as skin abrasions occur during space ﬂight, an enhanced Pseudomonas aeruginosa systemically or Klebsiella pneumoniae by inﬂammatory response in the setting of reduced immune compe- inhalation. Numbers of bacteria that allow most to all of the un- tence due to Treg migration to lymphoid organs could result in a treated animals to survive were used in these studies so that any reduced ability to control an infection.
decrement in immune function could be measured by increasedamounts of bacteria in the blood and lung and morbidity. Three 2.2.3. Effect of SPE radiation and hindlimb suspension on immune different strains of mice were used: ICR mice are an outbred strain function measured by bacterial challenge initiated in 1948 from Swiss mice, C3H/HeN mice are inbred and Infections of the skin, eyes and respiratory tract are com- have no known defects of polymorphisms that impair DNA re- mon in astronauts: infections have been reported 13 times in the pair or the response to ionizing radiation, whereas Balb/c mice Apollo and 8 times in Skylab missions, and spacecrafts need to be have 2 different polymorphisms in DNA-dependent protein kinases equipped with numerous antibiotics for treatment of such infec- (DNA-PKcs) that mediate non-homologous end joining, which re- tions A number of bacterial sults in decreased, but not absent, function infections have been observed in astronauts during or soon after To measure the effect missions, with organisms that do not typically lead to such infec- of the HS stress and SPE-like radiation on the ability of differ- tions in healthy people. As one example, Pseudomonas aeruginosa, ent strains of mice to effectively clear a challenge with bacteria, which does not ordinarily infect healthy people, was identiﬁed as hindlimb suspended and/or irradiated mice were exposed 5 days the pathogen that caused a serious life threatening urinary tract in- later to Pseudomonas aeruginosa or Klebsiella pneumoniae. The mice A.R. Kennedy / Life Sciences in Space Research 1 (2014) 10–43 were followed daily for signs of systemic and pulmonary infec- likely to be less effective with repeated applications over long pe- tions. The results for all three strains of mice were comparable riods of time. Numerous infections have already been documented and indicated that the mice exposed to HS and SPE radiation failed in astronauts and are considered a major hazard for spaceﬂight; to control a challenge with Pseudomonas aeruginosa or Klebsiella some infections have been minor while others have been serious pneumoniae, which led to a high morbidity/mortality rate and life-threatening, including a debilitating dental infection and as illustrated in [data from (note: in an incapacitating urinary tract infection these studies, morbidity was the same as mortality). Either SPE radiation or HS alone had some effects on morbidity in these It is known that spaceﬂight conditions alter the gene ex- studies, but when combined, they led to almost complete mor- pression patterns, virulence and virulence phenotypes of bacterial bidity/mortality. Other studies in this series of experiments led pathogens with evidence of increased vir- to the following conclusions: 1) similar levels of morbidity were ulence under space ﬂight conditions; thus, longer space ﬂights are observed after the challenge with Pseudomonas bacteria in male likely to lead to considerably more serious immunological prob- and female mice, indicating a lack of gender/sex differences in lems than observed so far in the space program.
this effect, 2) a dose of 1.5 Gy of total body radiation impaired The other immunological issue of great importance to the space the ability of mice to control the bacterial challenge in a fash- program is the observation of additive or synergistic-adverse ef- ion similar to that observed for 2 Gy with little difference in the fects caused by SPE radiation exposure and simulated microgravity morbidity observed between these two dose groups, but the mor- conditions; interactive effects between SPE radiation and simulated bidity differences observed for the 1 Gy dose group compared to microgravity have been observed for various immunological pa- those from the control group were not statistically signiﬁcant, in- rameters at doses of radiation that could be received by astronauts dicating that the threshold dose for morbidity resulting from the during space travel [e.g., bacterial challenge is between 1 and 1.5 Gy, 3) the relative in- The results indicate that, under simulated creases in morbidity were similar for all 3 strains of mice, sug- microgravity conditions, the effects of a given dose of SPE radi- gesting that the polymorphisms in DNA-PKcs in Balb/c mice did ation can be considerably more severe than the effects observed not signiﬁcantly affect the response to a bacterial challenge, and 4) for the same dose of radiation in normal, control animals. These peripheral blood granulocyte counts were determined in C3H/HeN results suggest the possibility that, in the space microgravity en- mice challenged with Pseudomonas aeruginosa at time points prior vironment, the effects of a given dose of SPE radiation could be to HS and irradiation and before and after the bacterial challenge.
comparable to those observed for a signiﬁcantly higher dose of ra- In these studies, there was a reduction in the peripheral blood granulocyte counts observed post-irradiation, which was similar tothat described previously 2.3. Emesis The numbers of peripheralgranulocytes increased as expected after bacterial challenge in con- The early phase of the acute radiation syndrome, which is trol or irradiated mice, but the mice treated with HS, with or known as the prodromal syndrome, can include nausea, retch- without radiation, failed to elevate the blood granulocyte counts as ing, vomiting, diarrhea, and fatigue expected Similar blunting of the peripheral blood These effects often manifest within 1 to 72 hours post- granulocyte counts in response to systemic infection in HS animals irradiation at sub-lethal doses, with a latency time inversely corre- was also observed in Balb/c and ICR mice (Drew Weissman and lated with dose. Vomiting is the reﬂexive act of forcefully ejecting colleagues, unpublished data).
the stomach contents through the mouth by coordinated mus-cle contraction. Published clinical studies have demonstrated that 2.2.4. Summary of the effects of SPE radiation on the immune system patients receiving total body irradiation or upper-abdominal irra- The effects of SPE radiation on immune system parameters diation often show nausea, retching and vomiting as side effects are of great importance to the space program as they are poten- tially life-threatening at doses that could conceivably be received lation between retching and vomiting events has been established by astronauts during space travel. It has been observed that ex- in the ferret model Emetic posure to SPE radiation along with HS, with additional exposure responses to various pharmacological agents, cytotoxins and radi- to a bacterial challenge, leads to a very high level of morbidity, ation have been compared previously among humans and various which is equal to mortality, in the studies performed in mice.
animal species including nonhuman primates, dogs, cats, and fer- The bacterial challenge utilized bacteria known to be associated rets Ferrets are considered to be a useful species in with astronaut infections and are already part of the spacecraft emesis research especially for radiation and environment (Pseudomonas aeruginosa) or that are part of the nor- cytotoxic drug-induced emesis and data mal bacterial ﬂora of the mouth, skin and intestines (Klebsiella from the ferrets have been used by the Department of Defense to pneumoniae); the bacterial challenges utilized in the mouse stud- develop a mathematical model for the human emetic response to ies described above were performed with bacterial levels that are radiation Another advantage of the ferret non-toxic (or minimally toxic) to the normal control mice. Un- model is that the prodromal response appears at lower radiation der the conditions described above leading to morbidity/mortality doses and with an earlier onset time as compared to other species, in the mice, death could be prevented by treatment with an an- including humans Thus, we have chosen ferrets as an tibiotic (enroﬂoxacin). Enroﬂoxacin is approved by the FDA as a experimental model system to determine the effectiveness of pro- veterinary antibiotic (marketed by the Bayer Corporation under the tons at the energy, doses and dose rate ranges relevant to the SPE trade name Baytril). A similar antibiotic (ciproﬂoxacin) is in wide radiation exposures expected during space travel.
use in human populations. While the effectiveness of the antibi- In our studies performed with female descented Fitch ferrets otic is outstanding, the major problem associated with the use of aged 12 to 16 weeks, irradiation with 60Co γ -rays or 155-MeV antibiotics for bacterial infections in astronauts is that organisms protons at a high dose rate of 0.5 Gy/minute resulted in dose- resistant to the treatment can grow out in a very short period of dependent changes in the endpoints related to retching and vom- time [as short as a one-week period of time (Personal Commu- iting, such as the fraction of animals that retched or vomited, the nication, Drew Weissman, M.D., Ph.D.)]. Thus, it is expected that number of retching and vomiting events, the length of the latency in the exploration class missions of the future, the antibiotics are period leading to the ﬁrst retching or vomiting event and the dura- A.R. Kennedy / Life Sciences in Space Research 1 (2014) 10–43 tion between the ﬁrst and last retching or vomiting events lial cells were exposed to 20 Gy irradiation with a 6-MeV elec- A dose–response relationship was observed for ferret tron beam at a ﬁxed dose of 2.4 Gy/minute vomiting and retching at the high dose rate. The minimum radia- It has also been shown that in human peripheral blood tion doses required to induce statistically signiﬁcant changes in the mononuclear cells (PBMNCs) after irradiation with a 6-MeV elec- retching- and vomiting-related endpoints were 0.75 and 1.0 Gy, re- tron beam, TF was up-regulated, which led to a signiﬁcant in- spectively; thus, these values are considered the threshold doses crease in PBMNC-associated pro-coagulant activity over a time for radiation induced retching and vomiting in the ferret model.
period of 7 days post-irradiation. Increased cellular TF protein The RBE of the proton radiation at the high dose rate did not dif- concentration was observed up to 7 days post-irradiation, and fer signiﬁcantly from 1. Similar, but smaller and less consistent, microparticle-associated TF activity was increased signiﬁcantly 3 changes in the retching- and vomiting-related endpoints were also days post-irradiation as compared with the non-irradiated con- observed for ferrets irradiated with γ -rays and protons delivered trols. PBMNC-derived microparticles post-irradiation also initiated at the low dose rate of 0.5 Gy/hour. Since this low dose rate is the plasma clotting faster than microparticles derived from con- similar to a radiation dose rate expected during an SPE, these re- trols. The radiation induced TF expression and increase in proco- sults suggest that the risk of SPE radiation-induced vomiting is low agulability of PBMNCs and cell-derived microparticles may repre- and may reach statistical signiﬁcance only when the radiation dose sent a possible mechanism by which ionizing radiation enhances reaches 1 Gy or higher.
blood thrombogenicity . In a study per- Ferrets have also been used previously to study emesis induced formed with leukoreduced fresh-frozen plasma irradiated with by radiation with 60Co γ -rays 30 Gy of γ -rays, prothrombin time (PT), activated partial throm- 0.6-GeV/n 56Fe ions, neutrons and 200-MeV boplastin time (aPTT), thrombin time, antithrombin III, protein C, protons and the emetic re- protein S, vWF, ristocetin cofactor, plasminogen–α2-antiplasmin, sponse in ferrets was found to be dependent on the type and dose the coagulation factors ﬁbrinogen, FII, FV, FVII, VIII, F IX, FX, of radiation. High LET 56Fe particles and ﬁssion neutrons were FXI, FXII, FXIII, and activated factor XII (FXIIa), D-dimer, ﬁbrin comparable in their ability to produce emetic responses (retching monomer, thrombin–antithrombin complex, prothrombin fragment or vomiting) in ferrets with an ED50 of 0.35 Gy and 0.40 Gy, re- 1 + 2 (F1+2), plasmin–α2-antiplasmin complexes, and platelet spectively whereas γ -rays were shown to be factor 4 were determined, and PT, aPTT and FVIII activities were intermediately effective with an ED50 of 0.77 Gy to found to be decreased signiﬁcantly, whereas activities of the co- 0.95 Gy and high energy electrons were the agulation factors FII, FV, FVII, FIX, FX, FXII were increased signiﬁ- least effective, with an ED50 of 1.38 Gy The cantly post-irradiation ED10, ED50 and ED90 values estimated for the fraction of animals To determine whether SPE-like proton radiation could affect that vomited after proton irradiation at the HDR were comparable blood clotting times, we have performed experiments with 12 to the ED10, ED50 and ED90 values after γ -ray irradiation at the to 16 week old descented female Fitch ferrets and demonstrated high dose rate in our study or at a dose-rate signiﬁcant increases in the PT at 3 hours post-irradiation with of 1 Gy/minute, as reported previously The ED10 and doses of 1 or 2 Gy (but not 0.25 Gy) of 110-MeV protons de- ED50, but not the ED90, values estimated for the fraction of animals livered at a high dose rate of 0.5 Gy/minute or 0.25 and 1 Gy that retched (or vomited) post-irradiation with protons at the HDR (but not 2 Gy) of 110-MeV protons delivered at a low dose rate were lower than the lower limits of the respective 95% conﬁdence of 0.5 Gy/hour Human PT values are com- intervals previously reported for γ -rays suggesting monly reported as an INR which is deﬁned as the patient's ‘test' that HDR proton irradiation was more effective than HDR γ -ray PT value divided by the laboratory ‘normal' PT value, raised to irradiation in inducing retching and vomiting.
the power of the International Sensitivity Index. INR values werecalculated for animals exposed to 2 Gy of 110-MeV protons at 2.4. Effects of radiation on blood coagulation and the development of both the high and low dose rates and 1 Gy of 110-MeV protons disseminated intravascular coagulation at the low dose rate, which resulted in the greatest PT responseto proton radiation in the study. Three out of 10 animals ex- Relatively little information exists in the literature on the ef- posed to 1 Gy at the low dose rate had an INR value of 2.0 fects of radiation on blood coagulation. Blood coagulation involves and an additional 3/10 of the ferrets had borderline INR values multiple components, which generate a ﬁbrin-rich blood clot to (> 1.75) approaching 2.0 which is con- stop bleeding in a process known as hemostasis. Primary hemosta- sidered to be of clinical concern for humans sis starts with the activation of platelets at the wound site by The INR values for the animals exposed to 2 Gy pro- exposing collagen to blood, which allows von Willenbrand fac- tons at the high dose rate were signiﬁcantly higher than the pre- tor (vWF) to bind to collagen and tethers platelets to the vas- irradiation levels, although they were still below 2. In addition to culature wall. The coagulation pathway, which is also referred the increase in PT, aPPT also increased 3 and 48 hours after 0.25, to as secondary hemostasis, occurs simultaneously on the nega- 1 or 2 Gy of 110-MeV proton irradiation at the low dose rate tively charged surface of activated platelets to generate a ﬁbrin-rich For the ferrets irradiated with 110-MeV thrombus The exposure of tissue protons at the high dose rate, signiﬁcant increases in aPPT was factor (TF) at the injury site of blood vessels, and its subsequent not observed in any of the radiation dose groups at 3 hours post- binding with Factor VII (extrinsic pathway), initiates a thrombin irradiation or in the 0.25 and 1 Gy dose groups at 48 hours post- (Factor II) burst that leads to ﬁbrin clot formation through the irradiation. The increase in PT induced by the proton irradiation at activation of a series of vitamin K-dependent serine proteases.
the high dose rate was due to Factor VII whereas Factors II, V, VII It is the activation of coagulation factors, such as Factor V and and IX contributed to the increases in PT induced by proton irra- VIII, by thrombin that drives the development of a stable ﬁbrin diation at the low dose rate These results clot as a part of the intrinsic pathway demonstrated that proton irradiation signiﬁcantly increased blood clotting times due to different coagulation factors, indicating po- Previous studies have demonstrated that radiation can induce tential radiation-induced coagulopathy. The ﬁnding that the effects vWF secretion from human umbilical vein endothelial cells irra- of the proton radiation at the low dose-rate are more severe than diated in tissue culture and the vWF mRNA those at the high dose-rate on an endpoint is an unexpected ﬁnd- levels were increased when either human or bovine endothe- ing in radiobiology, as the expectation is that reducing the dose A.R. Kennedy / Life Sciences in Space Research 1 (2014) 10–43 rate will have a sparing effect, thereby reducing the severity of ef- exposure to proton or γ -ray radiation produced step-wise changes fect on the biological endpoint being evaluated. Thus, the increased in hemostasis that begin with the radiation activated clotting cas- effect of low dose-rate irradiation on ferret blood clotting times is cade, which results in the cleavage of ﬁbrinogen and the formation of ﬁbrin clots. Activation of the coagulation cascade also leads to The blood clotting abnormalities in the ferrets are thought to increased PT and aPTT values due to the consumption of associated lead to a condition known as disseminated intravascular coagula- factors These changes occurred as early as tion (DIC), which is believed to result in 100% mortality in ferrets 3 hours post-irradiation, along with the detectable soluble ﬁbrin in irradiated with a 2 Gy dose of either γ -ray or SPE proton radiation the blood as well as detectable ﬁbrin clots in the blood vessels of The increases in the blood clotting times irradiated tissues In animals destined to die became more evident in ferrets destined to die from exposure to after irradiation, these abnormal hemostasis parameters became radiation. The LD50 dose for the development of DIC in ferrets is progressively worse. In ferrets exposed to a lower (sub-lethal) radi- ation dose (e.g., 1 Gy), the abnormal hemostasis characteristics re- While irradiation with SPE-like protons was shown to increase covered steadily and by day-30 post-irradiation, the clot formation, prothrombin time and partial thromboplastin time clot size, and platelet clumping values returned to baseline. These the mechanism for the proton radiation induced hypoco- results are particularly important, since the recognition of radiation agulation remains to be elucidated. We have hypothesized that induced DIC as a cause of death could change the course of actions the SPE-like proton irradiation activates the coagulation cascade, when the acute radiation syndrome is diagnosed and treated in which would put irradiated subjects in a hypocoagulable state. To people exposed to radiation through occupational accidents, radia- test this hypothesis, a separate experiment was performed with 12 tion terrorism or other catastrophic events to 15 week old descented female ferrets irradiated with 1 Gy of It is noteworthy that experiments performed as part of this 110-MeV protons at a dose rate of 0.5 Gy/hour, and the results in- project also documented the development of DIC in Yucatan dicate that the radiation exposure resulted in coagulation cascade minipigs (Krigsfeld, G.S., Shah, J.B., Sanzari, J.K., Lin, L. and Kennedy, activation, which was indicated by increases in soluble ﬁbrin con- A.R., Unpublished data). Three pigs were irradiated with a 2.5 Gy centration in the blood and ﬁbrin clots in blood vessels of livers, homogeneous dose of total body x-irradiation. One of these pigs lungs and kidneys from irradiated ferrets died, and another was euthanized; both of these pigs were diag- The soluble ﬁbrin concentration was determined using a rapid sol- nosed with DIC. The third pig did not die, but exhibited severe uble ﬁbrin assay that was previously developed and implemented blood clotting abnormalities (greatly increased bleeding times, at the Loma Linda University Medical Center to aid in early detec- etc.), as did the two pigs that did not survive. In addition, three tion of DIC in emergency room, operating room, or transplant pa- pigs were irradiated with a 2 Gy total body dose of SPE-like proton tients In addition to the activated coagulation radiation. These pigs exhibited severe blood clotting abnormalities cascade, PT and aPTT were also increased after irradiation, which is (increased blood clotting times), but they survived the radiation indicative of the involvement of the extrinsic/intrinsic coagulation pathways. The platelet counts in the irradiated ferrets remained 2.5. Effects of radiation on fatigue at approximately pre-irradiation values for up to 7 days post-irradiation, indicating that the observed effects on blood clotting Several experiments have been performed to evaluate the abil- times were not platelet-related. The activation of the coagulation ity of SPE proton and γ -ray radiation to induce fatigue in mice, cascade is expected to consume clotting factors, which, in turn, as reﬂected in changes in social exploration, submaximal exer- leaves the animal deﬁcient in clotting factors. Thus, the increased cise treadmill and locomotor activity PT and aPTT values in the irradiated animals might have been due The results of studies on social exploration indicated to radiation-induced effects on secondary hemostasis. WBC counts that low, but not high, dose-rate γ -ray and proton radiation expo- were reduced signiﬁcantly within 24 hours post-irradiation and sures led to comparable transient increases in social withdrawal, they remained reduced up to 7 days post-irradiation with a dose and these effects are thought to be due to a combination of re- of 1 Gy of SPE-like proton radiation.
straint stress and radiation. The results for studies on submaximal DIC is a serious, life-threatening condition in which clotting exercise treadmill indicated that neither γ -rays nor protons im- and bleeding are occurring at the same time, and it is often fa- paired performance on this test. In a study performed with 7 to tal due to multiple organ failure. Mechanistically, activation of the 8-week old male CD-1 mice irradiated with 0.5 or 2 Gy 137Cs clotting cascade is expected to decrease the bioavailability of the γ -rays at a dose rate of 44.5 cGy/minute (high dose rate), 60Co factors in the blood, thereby increasing the PT/aPTT values, as was γ -rays at a dose rate of 0.5 cGy/minute (low dose rate) or protons observed in the irradiated ferrets Ra- at dose rates of 0.5 Gy/minute (high dose rate) or 0.5 cGy/minute diation exposure may signiﬁcantly decrease leukocyte counts, and (low dose rate), locomotor activity was reduced in mice irradiated the prolonged low WBC, neutrophil, and lymphocyte counts can with γ -rays at the high dose rate but not in the mice irradiated leave the irradiated subjects at risk for infection, thereby further with γ -rays at the low dose rate or with protons, which had a overwhelming hemostasis and potentially leading to DIC, as has similar macroscopic dose distribution as that from 60Co γ -ray ex- been observed in patients with sepsis. Currently, the mechanism posure, when delivered at either the high or the low dose rate of radiation-induced death at the dose expected to kill 50% of the The γ -ray irradiation also increased hippocam- irradiated subjects (LD50) is thought to be due to bone marrow cy- pal TNF-α expression, which occurred as early as 4 hours post- totoxicity (known as the hematopoietic syndrome), which results irradiation and was followed by subsequent increases in IL-1RA in in a dramatic reduction in the number of circulating hematopoietic the cortex and hippocampus and reductions in activity-regulated cells and the resultant symptoms of infection (from white blood cytoskeleton-associated protein (Arc) in the cortex. These observa- cell loss) and bleeding (presumably from platelet loss) tions indicate that low-dose ionizing radiation rapidly activates the However, studies performed with ferrets have sug- neuroimmune system, potentially causing early onset fatigue-like gested that the death of the animals irradiated at the LD50 dose is symptoms in the irradiated animals.
due to a consumptive coagulopathy, which is followed by the on- In these studies related to proton or photon induced fatigue, set of DIC, since hypocoagulation occurred during early time points the results were consistent with a threshold effect, i.e., once a post-irradiation when the platelet counts were at normal levels dose suﬃcient to produce a response is given, additional dose does The ferret study results have shown that not increase the magnitude of the response. The magnitude of the

A.R. Kennedy / Life Sciences in Space Research 1 (2014) 10–43 Fig. 7. Loss of blood vessels in the pig dermis after a high skin dose exposure to SPE-like radiation. Pig skin tissue samples were taken pre-irradiation (Panel A) and at 30
days after the pig was exposed to a 10 Gy skin dose of 6 + 12 MeV electron radiation (Panel B). For the skin tissue taken before irradiation, vascular beds are denoted by
squares (Panel A). A consequence of exposure to the high skin dose from SPE radiation is that blood vessels disappear in the areas beneath the epidermis (Panel B), such
that the blood ﬂow to the cells in this area is reduced. Such areas of reduced blood supply beneath the epidermis occur in interventional radiology patients, and are known
to be life-threatening Thus, the areas with reduced blood supply must be removed surgically.
response with γ -rays is small, and additional stressors, such as Denuded vasculature, isolated from untreated and irradiated ani- restraint or other manual manipulations, are suﬃcient to obscure mals, was unresponsive to BK treatment, conﬁrming that the BK any acute behavioral changes. For those tests in which altered response is mediated via receptors present on the surface of en- behavior was noted for γ -rays, protons showed a similar trend, dothelial cells The fact that the relaxation which did not reach statistical signiﬁcance. Therefore, the RBE for response was lower in irradiated tissue suggests that radiation ex- proton radiation on this endpoint is assumed to be less than 1, al- posure damages the vascular endothelium. The results of this study though it could not be deﬁned mathematically due to the lack of a demonstrate that radiation has a direct effect on the cardiac vas- statistically signiﬁcant trend in the proton irradiated animals.
culature. Intact arteries from hearts taken from irradiated pigs ex- The data gathered on the ability of SPE radiation to induce hibited a reduced contractile force following exposure to BK. BK is fatigue in mice suggest that exposure to low-dose rate ionizing an endothelium-dependent dilator that acts directly on endothe- radiation leads to a minimal increase in fatigue, in the form of lial cells, causing them to release nitric oxide, PG12 and perhaps depressive/anxious behaviors, and that these effects are transient endothelium derived dilating factor(s), which signal relaxation of with full recovery within the 24 hour period vascular smooth muscle Our results suggest These behaviors are likely of equal or lesser magni- that alterations in vascular function are primarily a consequence tude than the depressive/anxious behaviors that are stimulated by of radiation-induced damage to endothelial cells, and that astro- the restraint stress necessary to perform the experiments and that nauts exposed to a high dose of SPE radiation could be at risk for is likely to be experienced by astronauts in typical space explo- vascular damage in the heart. While the skin dose was high in this ration vehicles. Thus, acutely, SPE radiation up to a 2 Gy total body study, the heart dose was only 0.35 Gy. This heart dose is consider- dose is highly unlikely to increase fatigue or other adverse behav- ably lower than the doses of radiation (0.5–5 G) previously shown iors over and above baseline levels for astronauts and is therefore to affect endothelial dependent relaxation in rat aorta highly unlikely to lead to mission critical fatigue.
2.6. Heart functional changes 2.7. Skin effects As part of the experiments designed to determine whether a The skin effects in pigs have been described previously high skin dose from SPE radiation has adverse effects on the in- and the immunological changes in pig skin accom- ternal organs of pigs, we evaluated changes in the heart brought panying the skin changes are described above in the section on about by a high skin dose of electron radiation planned to simulate immunology effects. It has been observed that pigmentation in- a dose distribution pattern like that expected from SPE radiation.
creases with increasing exposure to SPE radiation As changes in the left anterior descending (LAD, interventricular) and a method of quantitation for the melanin changes in artery can have major effects on heart function, our studies in irradiated pig skin has been developed (Billings, P.C., Sanzari, J.K., this area of research focused on LAD function in the pig hearts.
Kennedy, A.R., Cengel, A.K. and Seykora, J.T., Unpublished data). With The LAD artery provides the blood supply to the mid-region of very high skin SPE radiation doses, there is evidence of blood ves- the heart and is a major site of vessel stenosis. Vessels from con- sel loss in the pig dermis, as illustrated in A consequence trol and pigs exposed to electron radiation exhibited a similar of this radiation effect is reduced blood ﬂow to these areas of the relaxation response following treatment with adenosine diphos- dermis. It is known that areas of the dermis receiving a reduced phate and sodium nitroprusside. There was a reduced relaxation blood ﬂow can develop into a "diabetic foot" like state (which response to bradykinin (BK) treatment in the arteries from hearts needs surgical removal) in patients undergoing interventional radi- exposed to SPE-like electron radiation. In contrast, vessels obtained ology procedures In humans, the from control animals exhibited a 20% higher relaxation response development of such lesions requiring surgical removal takes ap- with BK, compared with arteries obtained from the irradiated pigs.
proximately one year or more and the skin doses associated with A.R. Kennedy / Life Sciences in Space Research 1 (2014) 10–43 such lesions are high (10 Gy) Such lesions could Upon evaluation of the optic nerve area, the meningeal sheath conceivably be a problem during the multi-year exploration class area was dilated in 31% of the irradiated animals, and the dif- missions planned for the future.
ferences between the measured area of dilation in the irradiatedpigs compared to the non-irradiated pigs were statistically sig- 2.8. Increased intracranial pressure and effects potentially related to niﬁcant. Further, an accumulation of lymphocytes, plasma cells, vision abnormalities evaluated in Yucatan mini-pigs and macrophages around the vessels near the optic nerve wasobserved, indicating an inﬂammatory response to radiation expo- Vision changes, characterized as a degradation in distant and sure; however, the presence of inﬂammatory inﬁltrates was not near visual acuity, have been documented in numerous astronauts consistent in the irradiated animals (inﬂammatory inﬁltrates were who have been involved in long-duration (of six months or longer) present in 10% of the irradiated pigs).
space ﬂight Although the etiology of the vi- The results described above suggest that SPE radiation may re- sion alterations is unknown, Mader et al. hypothesized that the sult in radiation-induced retinal atrophy or degeneration; however, optic nerve and ocular changes observed in astronauts could have long term studies will be necessary to determine whether the loss been caused by prolonged microgravity exposure of photoreceptor cells and changes in retinal width or optic nerve Since radiation exposure in clinical radiotherapy patients area persist at later time periods.
is known to be associated with increased intracranial pressure,increased radiation exposures during space travel could also con- 2.8.2. Ocular ultrasound results of the eyes of the pigs exposed to SPE tribute to the alterations thought to be involved in producing the vision abnormalities. We have evaluated the ability of SPE- Noninvasive measurements such as those obtained from bed- like radiation in pigs to produce changes like those associated side ocular ultrasound examinations have been advocated and uti- with the astronaut vision alterations, by performing ocular ultra- lized in the clinical setting and during spaceﬂight to give informa- sound examinations, lumbar puncture opening pressure studies tion about changes potentially related to vision abnormalities. Live and histopathologic examinations of eye tissue taken from pigs ir- ocular ultrasound imaging was performed in the pSPE animals ap- radiated with SPE-like radiation (in the forms of simulated electron proximately 2 months post-irradiation as part of a collaborative (6 + 12 MeV) SPE (eSPE) or simulated proton SPE (pSPE) exposures study between the investigators at the University of Pennsylvania The dosimetry studies have indicated that (Penn) and Wyle/NASA investigators skilled in ocular ultrasound the eyes and lenses receive substantial doses of radiation during examination techniques. The sonographer at Penn was remotely- an SPE; the dose to the lens is roughly comparable to the dose guided by an expert in eye/optic nerve imaging, and the remotely received by the skin of pigs (e.g., see guided sessions were conducted in a way that was similar to the"telemedicine" and "telescience" arrangement routinely used dur- 2.8.1. Histopathology changes in the eyes of pigs exposed to simulated ing ultrasound imaging sessions on the International Space Station proton and electron SPE radiation which could be related to vision (ISS). The optic nerve sheath diameter was measured using elec- tronic calipurs at three distances from the vitreoretinal interface.
The retina is a layered structure of neurons interconnected by Animals exposed to 5–10 Gy pSPE radiation exhibited a dose- synapses sending information to the brain via the optic nerve. The dependent trend in increased diameter measurements compared retina includes millions of photoreceptor cells, known as rods or to the non-irradiated animals. The measurements recorded for the cones, which are sensitive to light. Change or damage to the retina animals exposed to 5 Gy pSPE radiation resulted in a signiﬁcant can cause loss of vision. Signs of damage to the retina are sudden increase (p = 0.02 for right eye; p = 0.05 for left eye), compared ﬂashes of light, ﬂoating spots, decreased vision, or distorted vision.
to the non-irradiated group of animals. The 10 Gy dose pSPE ra- The ocular histopathology of pigs exposed to pSPE (5–10 Gy) or diation resulted in signiﬁcantly increased sheath diameters as well eSPE (5–20 Gy) radiation was examined. In this study, (Sanzari, J.K., (p = 0.002 for right eye; p = 0.03 for left eye) (Sanzari, J.K., Sar- Zeiss C.J., and Kennedy, A.R., Unpublished data) sagittal sections of gysan, A.E., Ebert, D., Garcia, K.M., Shultz, S.M., Seghal, C.M., and the entire eye and cross sections of the optic nerve were prepared Kennedy, A.R., Unpublished data). Ocular ultrasound examinations and qualitative differences (as well as some quantitative measure- were not performed on the corresponding animals exposed to eSPE ments) between the eyes of the irradiated and non-irradiated ani- radiation (for which histological changes are reported above).
mals were investigated.
When considered together, the histopathology and the ocular It was determined that eSPE radiation exposure resulted in a ultrasound exam results indicate that SPE radiation exposure di- decreased total retinal width in 20% of the irradiated animals com- rectly affects the eye structure of animals exposed to 5–20 Gy pared to the non-irradiated control animals; the differences ob- electron simulated SPE radiation or 5–10 Gy proton simulated SPE served in these studies between the irradiated and control pigs radiation. The observed changes in the optic nerve area may be were statistically signiﬁcant. Changes in retinal width can cause associated with the optic disc edema observed during/post space- turbulence in blood ﬂow and may indicate atrophy affecting the ﬂight reported by nerve ﬁber layer, ganglion cell layer, outer plexiform layer, and in-ner nuclear layer of the retina. It was conﬁrmed that the width of 2.8.3. Opening pressure in pigs exposed to proton and electron SPE the outer plexiform layer was also reduced in some of the animals exposed to eSPE radiation. A reduction in the width of the outer The opening pressure of pigs exposed to either pSPE or eSPE nuclear layer is an indicator of photoreceptor cell loss, which was was measured by lumbar puncture procedures and was found to conﬁrmed by the extrusion of photoreceptor nuclei in the retina.
be increased in some of the irradiated animals, with relatively The extrusion of photoreceptor nuclei (an indication of active cell larger increases in animals exposed to larger electron or proton SPE death) into the inner segments was observed in both the eSPE and radiation doses Pigs exposed to lower skin pSPE animals. The dose–response relationship was evaluated for doses of 2.5 to 7.5 Gy eSPE radiation (like the SPE radiation occur- the extrusion of photoreceptor nuclei induced by pSPE radiation ring in October 1972) exhibited increased opening pressure values, exposure and results indicated a statistically signiﬁcant slope, es- which lasted up to 90 days post-irradiation (i.e., at the time when tablishing that the loss of photoreceptor nuclei in pSPE irradiated the experiment was terminated), suggesting that SPE-like electron pigs was dose-dependent.
radiation resulted in increased intracranial pressure after a radia- A.R. Kennedy / Life Sciences in Space Research 1 (2014) 10–43 tion exposure with a skin dose as low as 2.5 Gy to 3 Gy protons (p < 0.001) or 0.5 Gy 56Fe ions (p < 0.05) than in mice receiving only the sham radiation The results in this area of research indicate that exposure to The decreased survival in the irradiated mice was accompanied even relatively low skin doses of SPE radiation can result in some by a signiﬁcant increase in the rate of development of malignant of the alterations thought to play a role in astronaut vision alter- lymphoma and Harderian gland tumors as well as the fractions of ations (e.g., increased intracranial pressure and increases in nerve animals with malignant lymphoma or rare tumors sheath diameter). Thus far, astronauts have not been exposed to signiﬁcant doses of SPE radiation during spaceﬂight, but it is ex-pected that there will be an increased risk of astronaut exposure to 3.2. Cataract development higher doses of SPE radiation in the exploration class missions ofthe future. It is hypothesized that exposure to SPE radiation along In the long-term experiment with CBA/JCR HSD mice exposed with extended space travel could exacerbate the development of to 1-GeV protons at a dose of 3 Gy or 1-GeV/n 56Fe ion radiation visual changes in astronauts. However, larger numbers of exposed at a dose of 0.5 Gy, the mice were observed daily over approxi- pigs than those used in the studies described above will be neces- mately two years after the radiation exposure. The animals were sary to conﬁrm and verify this hypothesis.
then euthanized and lenses were harvested and characterized us-ing an established classiﬁcation system that assigns discrete scores 2.9. Short-term survival in irradiated animals based on the severity of the lens opaciﬁcations. The results showedthat exposure to 1-GeV/n proton (3 Gy) or 56Fe ion (0.5 Gy) radi- In experiments performed with male ICR mice aged 4 to 5 ation signiﬁcantly increased the cataract prevalence and severity weeks exposed to 5.9, 6.8 or 7.2 Gy of total body irradiation with in CBA/JCR HSD mice to levels above the baseline levels of age- 1-GeV protons at dose rates ranging from 0.2 to 0.7 Gy/minute, induced cataract formation in this mouse strain the 30-day survival was 60%, 13.3% and 0%, respectively, and thecalculated lethal dose to kill 50% of the irradiated animals was 3.3. Cancer development 6.23 Gy In a separate experiment performedwith male ICR mice aged 4 to 5 weeks irradiated with 6 or 8 Gy Malignancy is considered to be a particular risk associated with of 225 kVp X-rays, the 30-day survival was 100% and 6.7%, respec- exposure to the types of ionizing radiation encountered during tively these survival levels are higher than space ﬂight. In these studies, the ability of protons and highly those observed in the 30-day survival studies in the animals ex- energetic, heavy particles (HZE particles) to induce carcinogene- posed to 5.9–7.2 Gy of 1-GeV/n protons. These results indicate that sis was determined in CBA mice. The major ﬁnding of the studies 1-GeV proton radiation is more lethal to mice than X-rays. Similar was that there was an increased risk of developing malignant lym- studies were performed by other investigators in C57BL/6J mice to phoma and rare tumor types, including Harderian gland tumors, in determine the relative toxicity of HZE radiation (1-GeV/n 56Fe ions) animals exposed to space radiations compared to γ -rays or 1-GeV protons In these A signiﬁcant increase in pre-malignant and malignant lesions of studies, the LD50/30 values for 56Fe ions, protons and γ -rays were myeloid origin was also observed in mice exposed to 3 Gy pro- reported to be 5.8, 6.8 and 7.25, respectively; the RBE value for the ton radiation and 0.5 Gy 56Fe ion radiation. These results indicate 56Fe ions was 1.25 and for the protons, the RBE was 1.06. It was that exposure to space radiations can increase the incidence of concluded from these studies that 56Fe ions caused accelerated and malignant tumors as well as pre-malignant lesions in mice. These more severe hematopoietic toxicity. Of interest in this study was studies have been reviewed recently the ﬁnding that intestinal crypt cells did not show increased HZEtoxicity. In another reported study performed in C57BL/6J mice, it 4. Radiation induced changes in gene expression
was observed that the LD50/30 values for 28Si and 12C ions were5.17 and 7.34 Gy, respectively In these stud- In a study performed with 5–6 weeks old ICR mice, irradia- ies, the RBE values for 28Si and 12C ions (compared to the γ -ray tion with 1-GeV protons was shown to increase the mRNA levels data in which the LD50/30 was 7.25) were 1.4 and 0.99, respec- for Bax, caspase-9, caspase-8, NFκ B1 and TGFβ1 and protein levels for Bcl2 and Bcl-xL The proton irradiation In studies performed with ferrets, the observed 30-day survival was also shown to induce cleavage of pro-apoptosis proteins, such was 100% for ferrets irradiated with up to a 1 Gy dose of SPE-like as caspase-3 and PARP-1, in bone marrow lysates of the irradiated protons and zero for ferrets exposed to 2 Gy of SPE-like protons animals. These results conﬁrm the ﬁndings that the high-energy The LD50/30 for SPE-like proton radiation proton radiation can induce the gene expression of classical mark- in ferrets was estimated to be approximately 1.5 Gy ers of apoptosis, as well as the downstream effectors, caspase-3 The survival curves for γ -ray irradiated ferrets were and PARP-1. In a separate study performed with ICR mice irradi- comparable to those for the proton-irradiated animals, and both ated with 1.0 and 6.4 Gy of 1-GeV protons or 1.1 and 7.0 Gy of the proton and γ -ray irradiated ferrets displayed signs of distress γ -rays to compare the acute effects of radiation on gene expres- including ecchymosis, petechiae, and hemorrhaging. It was hypoth- sion in radiation-sensitive tissues (e.g., spleen, thymus, bone mar- esized that the ferrets were dying of DIC in these studies row, testis and the GI tract), the apoptotic responses were found which was conﬁrmed by additional evidence to vary greatly between γ -ray and proton irradiated animals in a tissue- and dose-dependent manner and cell death in the splenicwhite pulp was consistently lower in the proton-irradiated animals 3. Long-term effects
compared to the γ -ray irradiated animals Both proton and γ -ray irradiation triggered nuclear accumulation 3.1. Long-term survival in irradiated animals of p53, with no signiﬁcant differences in the majority of the knownpro-apoptotic p53-target genes in the spleens of irradiated mice.
In a long-term study in which male CBA/JCR HSD mice aged 8 However, γ -ray irradiation uniquely triggered a pro-apoptotic ex- to 9 weeks were irradiated with 3 Gy of 1-GeV protons or 0.5 Gy pression proﬁle in the spleen and Peyer's patches, which exhibited of 1-GeV/n 56Fe ions and monitored for 2 years after irradiation, a higher level of apoptosis after γ -ray irradiation than after the the survival of the mice was signiﬁcantly lower in mice exposed proton irradiation despite the increased presence of DNA strand A.R. Kennedy / Life Sciences in Space Research 1 (2014) 10–43 breaks and phosphorylated-ATM in the spleens of proton irradi- cell membrane components, antioxidants could be effective coun- ated animals. Differences in the acute pro-apoptotic response to termeasures against radiation induced oxidative stress and other proton and γ -ray irradiation correlated with increased expression adverse biological effects occurring downstream to the free radical of the p53-dependent pro-apoptotic gene, Bcl-G, and granzyme B, suggesting that the fate of the cells after proton and γ -ray irra- The agents evaluated in our studies as potential countermea- diation may be context-dependent and the triggering of apoptosis sures for radiation induced oxidative stress included N-acetyl cys- in lymphoid cells after irradiation may not be dependent solely on teine (NAC), ascorbic acid (or vitamin C), coenzyme Q10, folic acid, the extent of DNA damage brought about by the radiation expo- glutathione, α-lipoic acid, niacin, L-selenomethionine (SeM), thi- amin and vitamin E succinate. NAC is a small molecular weight In female ICR mice irradiated with 60Co γ -rays at low thiol and a precursor to intracellular cysteine and glutathione (0.5 Gy/hour) or high (0.5 Gy/minute) dose rates, changes in the which is a tripeptide small molecular weight thiol expression of genes implicated in oxidative stress, extracellular shown to be a versatile protector against radiation induced ox- matrix (ECM) remodeling and selected protein expression proﬁles idative damage NAC is also effective in mouse skin were examined using skin tissues harvested at 4 in activating NF-κB and manganese superoxide dismutase (Mn- hours post-irradiation After irradiation at doses SOD) gene expression as low as 0.25 Gy, the expression of many genes responsible for MnSOD is a main mitochondrial antioxidant enzyme with ra- regulating the production of reactive oxygen species were signif- dioprotective properties icantly altered by more than 2-fold as compared to unirradiated Vitamin C is a water-soluble antioxidant that controls. The expression proﬁles of 18 to 20 of the 84 ECM genes reacts with highly damaging hydroxyl radicals to form less toxic were also signiﬁcantly altered after irradiation at the low dose ascorbate free radicals, which can be detoxiﬁed by enzymes that rate. As compared to the low dose rate irradiation, the high dose reduce ascorbate free radicals back to ascorbic acid rate irradiation resulted in different ECM gene expression proﬁles, Vitamin E is a lipophilic agent that protects cell membranes from with the most striking differences observed for genes encoding oxidative damage by radiation or other physical or chemical agents matrix metalloproteinases. These results indicate that the expres- Dietary supplementation of vitamins C and E sion of many genes involved in oxidative stress responses and ECM is thought to be important for protection against human diseases remodeling may be differentially regulated by high and low dose associated with free radical damage to cellular DNA, lipids and pro- rate irradiation.
teins Lipoic acid is a B vitamin that is bothlipid and water-soluble and is considered to be a "universal antiox- 5. Countermeasures and mitigation of space radiation damage
idant" because it can react with hydroxy radicals, singlet oxygen,and peroxyl and hypochlorous radicals It is anexcellent radical scavenger both in the oxidized and reduced form It has been well established that the biological effectiveness of and is known to regenerate other antioxidants from their inactive ionizing radiation depends on the LET, which describes the rate of forms. As examples, lipoic acid plays an essential role in mito- energy loss along the trajectory of ionizing particles chondrial dehydrogenase reactions It also protects and the ion species cell membranes by reacting with and regenerating vitamin C and Ionizing radiation damages cells through a combination of direct glutathione, which in turn recycle vitamin E action, which refers to the direct hit of biologically important Treatment with lipoic acid has been shown to reduce radiation- targets by the particle radiations, and indirect actions via water- induced oxidative stress derived free radicals produced by the radiation and hematopoietic tissue damage in irradi- While the indirect action plays an important role in the bi- ated mice Treatment with lipoic acid ological effects of low-LET radiations, such as X-rays and γ -rays, in combination with vitamins C and E has been shown to pro- its contribution diminishes with increase in LET and the direct ac- tect against lens damage caused by low dose irradiation tion contributes more to the biological effects of high-LET radiation Selenium is an essential trace element for main- than the indirect action taining activities of the important antioxidant enzymes, thiore- From our studies, there have been many publications related doxin reductase and glutathione peroxidase to countermeasures for acute radiation effects Together with vitamin E, it pro- tects cell and organelle membranes from oxidative damage, facil- itates the union between oxygen and hydrogen at the end of the metabolic chain and the transfer of ions across cell membranes These publications are Another agent evaluated as a potential brieﬂy described below.
countermeasure for radiation induced oxidative stress is a soybean-derived protease inhibitor known as the Bowman–Birk inhibitor 5.1. Radiation induced oxidative stress and antioxidants as (BBI), which has been developed in the form of BBI Concentrate (BBIC) for cancer prevention and human trials, as reviewed pre-viously Both BBI and BBIC Using a dichloroﬂuorescein (DCF) ﬂuorometric assay that we have been shown to have antioxidant properties had previously adapted and standardized to measure radiation in- and BBI has been utilized as a radioprotective agent duced oxidation in live cells we have demonstrated that low LET photon radiation, such as X- rays and γ -rays, and high LET radiation, such as 0.6-GeV/n silicon Using the DCF ﬂuorometric assay method previously adapted ions and 1- or 5-GeV/n 56Fe ions, as well as 250 MeV protons, are for measurement of radiation induced oxidative stress in cultured all capable of inducing oxidative stress sug- cells we performed experiments with X- gesting that the indirect actions via free radical generation may rays, γ -rays, protons and HZE particles to evaluate the protective contribute substantially to the biological effects of both low and effects of antioxidants against radiation induced oxidative stress high LET radiation. Since the removal of radiation generated free and found NAC, ascorbic acid, α-lipoic acid and SeM to be highly radicals will make them unavailable to damage DNA, proteins and effective in preventing radiation induced oxidative stress, whereas A.R. Kennedy / Life Sciences in Space Research 1 (2014) 10–43 coenzyme Q10 and vitamin E succinate were only weakly effective determined in HTori-3 cells by the soft agar colony formation as- in preventing radiation induced oxidative stress in cultured cells say, which measures the capability of HTori-3 cells to grow in Based on the results of an anchorage-independent manner. The results indicate that treat- the DCF ﬂuorometric assay experiments, these antioxidants were ment of the cells with BBI, BBIC, SeM alone or in combination with selected as a combination for further studies in vitro and in vivo.
ascorbic acid, coenzyme Q10 and vitamin E succinate prevented The ability of the antioxidant combination and BBIC to pre- proton and HZE particle radiation induced HTori-3 cell transforma- vent radiation induced oxidative stress in vivo was evaluated in tion in vitro In experiments performed Sprague-Dawley rats and CBA mice irradiated with γ -ray, proton or with γ -ray radiation, treatment with 5 μM SeM before, during 56Fe ion radiation using the total antioxidant status (TAS) in serum and/or as late as 7 days after the radiation exposure brought the or plasma as the biological endpoint anchorage-independent colony formation eﬃciency down to levels In the rat studies, the serum or plasma that were not signiﬁcantly different from the sham radiation con- level of total antioxidants was found to be decreased after expo- sure to γ -ray or 1-GeV/n 56Fe ion radiation, and the decrease wasalleviated or completely prevented in the animals fed with a diet 5.3. Antioxidant protection against space radiation induced mortality supplemented with SeM (12 μg/g diet) alone or in combinationwith sodium ascorbate (19 μg/g diet), NAC (51 μg/g diet), α-lipoic It is well known that the hematopoietic system is highly sen- acid, reduced form (100 μg/g diet), vitamin E succinate (8.6 μg/g sitive to total body irradiation (TBI) and the fate of hematopoietic diet) and coenzyme Q10 (51 μg/g diet) cells after TBI may determine the survival or death of irradiated In the mouse studies, the plasma TAS also subjects Thus, we decreased signiﬁcantly after exposure to 0.5 Gy of 1-GeV/n 56Fe have evaluated the effects of antioxidants in mice using the 30-day ion radiation or 3 Gy of 1-GeV proton or γ -ray radiation, and survival level and hematopoietic cell counts as the biological end- the decrease in plasma TAS was alleviated or prevented com- points. Dietary supplementation with an antioxidant combination pletely by diet supplementation with BBIC (10 mg/g diet), SeM consisting of SeM (0.06 μg/g diet), α-lipoic acid (85.7 μg/g diet), (0.14 μg/g diet), or a combination of L-SeM (0.14 μg/g diet), NAC (171.4 μg/g diet), sodium ascorbate (142.8 μg/g diet) and vita- sodium ascorbate (17.14 μg/g diet), NAC (51.43 μg/g diet), α-lipoic min E succinate (71.4 μg/g diet) signiﬁcantly improved the 30-day acid (102.86 μg/g diet), vitamin E succinate (8.57 μg/g diet) with survival of the mice irradiated with 8 Gy of X-rays or without coenzyme Q10 (51.43 μg/g diet) or 5.9 Gy of protons However, no sig- These results indicate that BBIC, SeM and niﬁcant improvement was observed for the mice irradiated with the antioxidant combinations are potentially useful as countermea- protons at higher radiation doses (6.8 or 7.2 Gy) sures against space radiation-induced oxidative stress and subse- It is expected that the higher proton radiation doses used quent adverse biological effects, which could arise from the in- in these studies might have caused damage that was beyond miti- creased oxidative stress in irradiated subjects.
gation by the antioxidant treatment. In both the proton and X-rayradiation experiments, antioxidants were more protective when 5.2. Antioxidant protection against radiation induced cell death and the antioxidant treatment was initiated 2 hours after radiation ex- transformation in vitro posure as compared to the antioxidant treatment initiated 7 daysprior to radiation exposure These re- The protective effects of the antioxidants, BBI and BBIC against sults were conﬁrmed by other investigators, who also showed that radiation induced cell death have been evaluated in vitro using the antioxidant combination had a better protective effect on radi- the clonogenic survival of cultured MCF10 cells or HTori-3 cells ation induced lethality when started 7 days post-irradiation than as the biological endpoints Irradi- it did when it was started 2 hours post-irradiation ation with 5-GeV/n 56Fe ions resulted in a dose-dependent de- The ﬁndings that antioxidant treatment leads to better sur- crease in the clonogenic survival of the MCF10 cells, which was vival when applied post-irradiation might result from an adaptive attenuated by treatment with SeM alone or in combination with response to radiation exposure, which has been reviewed recently ascorbic acid, coenzyme Q10 and vitamin E succinate with an es- The ability of the antioxidants to timated dose modifying factor (DMF) of 1.2, 1.4, 2.2 and 2.2, re- improve radiation survival even when the antioxidant treatment spectively, for BBI, BBIC, SeM alone or in combination with other was initiated after the radiation exposure suggests that antiox- antioxidants The identical DMF values for idants can be a feasible countermeasure for radiation exposure treatments with SeM alone or in combination with other antioxi- associated with space travel, radiation accidents or terrorist attacks dants indicate that these antioxidant combinations were not more in which radiation exposure could occur without much advanced effective than the SeM treatment alone under the experimental conditions utilized. These results do not rule out the possibil- The antioxidant treatment signiﬁcantly attenuated the radia- ity that the antioxidant combinations might have provided bet- tion effects on peripheral hematopoietic cell counts in the mice ter protection than SeM treatment alone under other experimen- irradiated with 1 or 8 Gy of X-rays or 1 tal conditions or for other biological endpoints, however, since or 7.2 Gy of 1-GeV protons The antioxi- a combination of antioxidants with different biochemical proper- dant treatment was also shown to improve the recovery of the ties and action mechanisms is likely to provide better protection bone marrow cell counts in mice irradiated with γ -rays for different molecular targets during and after irradiation. For or 1-GeV protons Thus, an- examples, cationic thiols are much more effective than anionic tioxidants appear to be effective for protection of hematopoietic thiols in protecting DNA against radiation damage cells against the adverse effects of either photon or proton radi- and lipophilic antioxidants, such as vitamin ation. The ability of antioxidants to prevent the radiation caused E, are effective protectors of biomembranes loss of circulating neutrophils, also called PMNs, are illustrated in whereas hydrophilic antioxidants are more ef- which shows that the mice maintained on an antioxidant fective in protecting soluble proteins and enzymes in the aqueous diet and exposed to an 8 Gy dose of radiation experienced a drop environment of cells in the levels of PMNs/neutrophils of a magnitude comparable to The protective effects of the antioxidants, BBI and BBIC, against the effects observed for mice exposed to a 1 Gy dose of radia- proton and HZE particle radiation induced cell transformation were tion and maintained on the normal diets. The mice maintained A.R. Kennedy / Life Sciences in Space Research 1 (2014) 10–43 or 0.5 Gy of 1-GeV/n 56Fe ions, treatment with the antioxidantcombination or BBIC decreased the fractions of animals with ma-lignant lymphoma to levels that were not signiﬁcantly differentfrom the baseline level The treatment withthe antioxidant combination or BBIC also prevented the increasein the fractions of mice with premalignant or malignant lesionsof myeloid origin after the proton irradiation and the incidencerate of rare tumors (which included Harderian gland tumors) af-ter the proton or 56Fe ion radiation exposures From these experiments, it was concluded that an-tioxidants have a major protective effect against space radiationinduced carcinogenesis in vivo Inthese studies, a major protective effect resulted from the abilityof the antioxidants to prevent the early stage neoplastic growthsfrom growing into fully developed, malignant tumors. Other stud-ies suggest that anticarcinogenic agents can be added at late timesfollowing carcinogen exposure, in both in vitro and in vivo sys- Fig. 8. Effects of radiation on polymorphonuclear (PMN)/neutrophil cell counts in
tems, and still have a suppressive effect on the carcinogenic pro- mice treated with antioxidants. In this experiment, animals were maintained on the cess The results of the studies on radiation control diet or antioxidant supplemented diet and irradiated with x-rays at doses of induced carcinogenesis suggest that antioxidant and BBIC supple- 1 or 8 Gy, which signiﬁcantly decreased the numbers of circulating PMN cells (neu-trophils) to a greater extent in mice maintained on the control diet than in mice ments could be useful for the prevention of malignancies and other maintained on the antioxidant supplemented diet. The magnitude of PMN cell loss neoplastic lesions developing as a result of exposure to space radi- in mice maintained on the antioxidant diets and exposed to 8 Gy of radiation was ation. It has been concluded from these studies that antioxidants comparable to that observed in the mice maintained on the control diet and ex- have a major protective effect against radiation induced carcino- posed to a 1 Gy dose of radiation. The decrease in the PMN cell counts in micemaintained on the antioxidant supplemented diet and exposed to a 1 Gy dose of genesis, and are effective even when added at late times during radiation was not signiﬁcantly different from that in the control animals. These re- the carcinogenic process.
sults indicate major beneﬁcial effects of antioxidants on the survival of circulatingPMN cells/neutrophils following the radiation exposure. [These data are representa- 5.6. Mechanism(s) for antioxidants as radiation countermeasures tive of those published previously To study the mechanisms for the radioprotection by the an- on the antioxidant diets and exposed to a 1 Gy dose of radia- tioxidant treatment, we have examined the expression of the ATR tion had PMN/neutrophil cell counts that were not signiﬁcantly gene, which is one of the central components of the DNA dam- different from those in the control animals. Thus, it is concluded age response pathway and the CHK2 that the antioxidant diet had highly signiﬁcant beneﬁcial effects on gene, which is a cell cycle checkpoint regulator and putative tumor PMN/neutrophil cell counts in irradiated animals.
suppressor in HTori-3 cells ir-radiated with 0.4 Gy of 5-GeV/n 56Fe ions with or without SeM 5.4. Antioxidant protection against space radiation induced cataracts (5 μM) treatment initiated 24 hours prior to the radiation expo-sure. The results indicate that the ATR mRNA level was increased An increased rate of cataract formation has been observed in by 42% with the SeM treatment alone and increased by 94% with astronauts which has the SeM treatment and radiation exposure been attributed to the increased exposure to cosmic radiation dur- In the same study, SeM treatment alone did not signiﬁcantly affect ing space travel. To investigate the ability of BBIC and antioxidants the CHK2 mRNA level, but the combined treatment with SeM and to reduce the formation and severity of cataracts related to space 56Fe ion radiation increased the level of CHK2 mRNA by 99%. The radiation exposure, mice were exposed to 1-GeV proton or 1-GeV/n up-regulation of ATR and CHK2 gene expression observed in the 56Fe ion radiation and fed with a control diet or diets supple- irradiated HTori-3 cells may prevent the cells from going through mented with BBIC or the antioxidant combination containing SeM, mitosis until the damage is repaired, thereby preventing the ra- NAC, ascorbic acid, coenzyme Q10, α-lipoic acid and vitamin E suc- diation damage from being ﬁxed and leading to mutations and/or cinate before and for 2 years after the radiation exposure Lenses were harvested approximately 2 years after In ICR mice irradiated with 6.4 Gy of 1-GeV protons or 7.0 Gy the radiation exposure for evaluation of the lens opaciﬁcations. The of γ -rays, 15 genes belonging to the class of "apoptosis regula- results showed that treatment with BBIC or the antioxidant com- tor activity" were differentially expressed in the spleen of mice fed bination decreased the prevalence and severity of the lens opaciﬁ- an antioxidant supplemented diet as compared with mice fed with cations in the mice irradiated with 3 Gy of 1-GeV proton or 0.5 Gy the control diet, and the antioxidant treatment inhibited apoptosis of 1-GeV/n 56Fe ion radiation, although statistical signiﬁcance was in the white pulp of the spleen following γ -ray irradiation, pos- only achieved for the 56Fe ion irradiated mice, possibly due to the sibly by altering IL-6 signaling and by blocking the expression of higher proton radiation dose (3 Gy) that might have exceeded the the prokineticin PROK2, the ligand to the G protein-coupled re- protective capacity of the antioxidant combination or BBIC. These ceptors PROKR1 and PROKR2 which are results indicate that BBIC and the antioxidant combination could involved in a number of pathophysiological processes. In ICR mice be useful for protecting astronauts against space radiation-induced irradiated with 1 or 8 Gy of X-rays, bcl-2 gene expression was cataracts during or after long-term manned space missions.
found to be decreased, whereas bax, caspase 7, caspase 9 andTGF-β1 gene expression was increased at 4 or 24 hours after irra- 5.5. Antioxidant prevention of space radiation induced cancer diation. Dietary supplementation with antioxidants attenuated theradiation effects on bax, caspase 7, caspase 9 and TGF-β1 gene Radiation induced malignancy is a particularly important risk expression, but increased bcl-2 gene expression by 10 fold at 24 associated with extended space travel. In a 2-year study performed hours after irradiation The abrogated pro- with CBA/JCR HSD mice irradiated with 3 Gy of 1-GeV protons apoptosis (bax and caspase 9) gene expression and the increased A.R. Kennedy / Life Sciences in Space Research 1 (2014) 10–43 anti-apoptosis (bcl-2) gene expression observed in the irradiated experiment performed with ferrets (aged 12–15 weeks), that were animals treated with antioxidants have implicated apoptosis as a exposed to a 2 Gy total body dose of γ -ray radiation with sub- key process modulated by antioxidants to attenuate the effects of cutaneous injection of phosphate buffered saline (PBS) or Neulasta radiation on the hematopoietic system and animal survival (0.1 mg/kg) on days 1, 4, and 7 post-irradiation, peripheral blood The anti-apoptotic effects of selenium have also been was collected from each animal on day 0 (prior to radiation ex- reported in normal human or mouse ﬁbroblasts posure), and on days 1, 4, 7, and 13 post-irradiation for analysis.
and primary human keratinocytes irradiated The results demonstrate that the Neulasta treatment of irradiated with ultraviolet irradiation. The anti-apoptotic effect of selenium ferrets could lead to a signiﬁcant increase in neutrophil counts observed in normal animals or tissues after irradiation is in con- (Krigsfeld, G.S., Sanzari, J.K. and Kennedy, A.R., Unpublished data). In a trast to the pro-apoptotic effect of selenium observed in malignant similar experiment performed with pigs (aged 12–15 weeks) that cell lines or tissues were exposed to a 2 Gy total body dose of SPE-like proton radi- The differential effects of selenium on apop- ation and subcutaneous injections of PBS or Neulasta (0.1 mg/kg) tosis in normal and malignant cells/tissues suggest the possibility on days 4, 7, and 10 post-irradiation, peripheral blood was col- that selenium may protect normal tissues against radiation dam- lected from each animal on day 0 (prior to radiation exposure), age without the unintended consequence of suppressing radiation and on days 1, 7, 10, 13, and 30 post-radiation for analysis. The induced apoptosis in malignant cells or tissues. The different ef- results indicated that the proton radiation exposure led to statisti- fects of antioxidants in normal cells, as compared to their effects cally signiﬁcant decreases in neutrophil counts at days 7, 10 and in malignant cells, or those in different stages of carcinogenesis, 13 post-irradiation in the irradiated animals injected with PBS; have been discussed elsewhere however, in irradiated pigs treated with Neulasta, the neutrophil In a separate study performed with cultured HTori-3 cells ex- counts were never decreased to below the baseline levels (Sanzari, posed to low doses (0.1 and 0.2 Gy) of 1-GeV/n 56Fe ion radiation, J.K., Krigsfeld, G.S., Shuman, A.L. and Kennedy, A.R., Unpublished data).
treatment with 5 μM SeM in the medium for 24 hours prior to ir- The overall conclusion from the studies in mice, ferrets and pigs is radiation profoundly affected the radiation induced alterations in that Neulasta can increase the number of circulating neutrophils in gene expression The exposure to 0.1 and 0.2 three different species of animals evaluated.
Gy of 56Fe ion radiation induced signiﬁcant differential expressionof 196 and 610 genes, respectively, and the differential expression 5.8. SI–Wu–Tang or fructose as countermeasures to increase neutrophil of 39% to 55% of these genes was abolished by the SeM treat- counts in mice exposed to SPE or γ -ray radiation ment Genes and functional pathways thatwere signiﬁcantly up- or down-regulated by the 56Fe ion irradi- A Chinese formula SI–Wu–Tang (SWT, ation in the absence, but not in presence, of SeM treatment have given to radiation and chemotherapy cancer patients in China to been summarized previously Of partic- mitigate the adverse effects of cancer therapy on the numbers ular interest was a cluster of chemokine and cytokine genes, e.g., of circulating blood cells. One of the major ingredients in SI– CXCL1, CXCL2, IL6, IL11, IL8, IL24 and TGFβ2, which showed in- Wu–Tang is fructose, as reviewed creased expression after irradiation with 0.1 Gy of 1-GeV/n 56Fe We have performed studies in mice to determine whether these ions in the absence, but not in the presence, of 5 μM SeM in the compounds in traditional Chinese medicine were able to mitigate medium before and during the radiation exposure reduced circulating blood cell counts following γ -ray or SPE-like It is also noteworthy that SeM has been shown to reduce proton radiation. The main conclusions of the studies were as fol- space radiation induced effects by mitigating stress-related signal- lows: 1) SWT and fructose were both capable of increasing the ing pathways and downregulating certain genes associated with number of circulating lymphocytes in γ -ray or SPE proton irra- cell adhesion These results suggest that diated mice, and 2) fructose was more effective than SWT in in- SeM is potentially useful as a countermeasure to prevent some of creasing the number of circulating lymphocytes in γ -ray or proton the acute inﬂammatory/immune responses induced by low-dose irradiated mice These results are HZE particle radiation.
important since there are essentially no countermeasures for lym-phocyte loss following radiation exposure. Lymphocytes are among 5.7. Granulocyte colony-stimulating factors as countermeasures the most sensitive cells in the body to radiation exposure , and a reduced In addition to the antioxidants, two forms of granulocyte lymphocyte count in the circulation at post-irradiation times is ex- colony-stimulating factors (G-CSFs), ﬁlgrastim and pegﬁlgrastim, pected to result in greater susceptibility to infections. The fact that were evaluated as countermeasures using the neutrophil count in both SWT and fructose can be given orally makes them attractive ICR mice irradiated with γ -rays or SPE-like protons as the biolog- for use during space travel.
ical endpoint. The results demonstrated that exposure to SPE-likeproton radiation or γ -ray radiation at doses up to 2 Gy signiﬁ- 5.9. Antibiotics as countermeasures for bacterial toxicity in mice cantly decreased circulating neutrophil counts in a dose and time exposed to SPE radiation and HS dependent manner, which was prevented by treatment with ei-ther form of G-CSF evaluated in the study Enroﬂoxacin, an antibiotic for veterinary use, was studied for its These results indicate that both forms of G-CSFs could be effectiveness as a countermeasure in experiments using the bacte- a potential countermeasure for the reduced number of neutrophils rial challenge model in mice. Enroﬂoxacin is available in an oral in irradiated animals, although pegﬁlgrastim appears to be supe- form but the mice in these experiments were treated with sub- rior since its stimulatory effect on the neutrophil count was more cutaneous drug. Control, irradiated (2 Gy), hindlimb suspended, pronounced and lasted longer than that of ﬁlgrastim.
and irradiated and hindlimb suspended mice were treated with In mouse studies using G-CSF (Neulasta) as a countermeasure Pseudomonas aeruginosa bacteria at a dose that can be cleared by for bacterial challenge toxicity in animals exposed to SPE radiation untreated animals or, in later experiments, with a dose of bacteria along with HS, Neulasta treatment was shown to reduce morbid- that leads to morbidity in approximately 50% of untreated mice.
ity from 80–90% to 20–30% in γ -ray or proton irradiated C3H/HeN With both challenge doses, the institution of enroﬂoxacin at the mice exposed to HS and challenged with Pseudomonas aeruginosa time of bacterial challenge reduced morbidity from 80–100% to bacteria (Drew Weissman and colleagues, Unpublished data). In an 0%. This demonstrated the complete effectiveness of prophylactic A.R. Kennedy / Life Sciences in Space Research 1 (2014) 10–43 antibiotic treatment in the protection of irradiated and hindlimb many of the pigs with this condition developed non- suspended mice against bacterial toxicity with the potential to lead productive coughs. Thoracic radiographs and diagnostic CT scans to morbidity/death (Drew Weissman, Unpublished data).
were performed, and the CT ﬁndings were consistent with an acute While the effectiveness of the antibiotic countermeasure cannot lung injury concurrent with chronic bronchial changes and vol- be surpassed, it does carry a number of potential adverse effects, ume loss. Differential diagnoses for these ﬁndings were radiation especially, if it will need to be used multiple times, including the induced lung injury or atypical infectious bronchopneumonia. To generation of antibiotic resistant bacteria. Thus, it is believed that help differentiate between these possibilities, sequential antibiotic alternative countermeasures for bacterial toxicity, with different therapy, which included atypical (e.g., mycoplasma), Gram-positive mechanisms of action than those of antibiotics, will be useful for and Gram-negative coverage, was initiated. After two weeks of space travel in future exploration class missions.
therapy, it was concluded that antibiotics were not effective treat-ments for the condition. Corticosteroid therapy then began and the 5.10. Countermeasures for radiation induced emesis in ferrets pig symptoms improved rapidly, with dramatic improvement andresolution of imaging abnormalities observed within one month.
In studies performed with ferrets, 5-HT3 receptor antagonists, It was concluded that corticosteroids were effective treatments for such as Zofran, have been shown to prevent or ameliorate vom- the pneumonopathy and/or pneumonitis that developed in the pigs iting and retching in ferrets following proton radiation exposure exposed to SPE-like radiation As Zofran is maintained on the ISS for nauseaand vomiting, it is the recommended 5-HT3 receptor antagonist to 5.13. Mometasone as a countermeasure for SPE proton radiation prevent or mitigate radiation induced nausea and vomiting during induced skin lesions in pigs space travel.
Pigs were exposed to either a 5 Gy or 10 Gy dose of SPE-like 5.11. Countermeasures for altered bleeding times in ferrets after SPE electron irradiation, and seven types of creams were applied to the trunk of the animal in 1 inch2 patches covered with Tega-derm dressing consecutively for 14 days immediately after the It is well established that endotoxin (lipid A portion) released radiation exposure. The only cream that appeared to mitigate by Gram negative bacteria activates certain factors of the intrin- the radiation-induced initial hyperpigmentation was the cream sic coagulation cascade, such as Factor XII, which in turn initiates that contained corticosteroids (mometasone cream [Elecon]), as ﬁbrin formation and increases PT/aPTT values, thereby increasing compared to the other creams, which were water-based emol- the risk of DIC development. To counteract the factor deﬁciencies, lients, a platelet growth factor containing cream, a triple antibi- BeneFIX (recombinant Factor IX) was evaluated as a countermea- otic containing cream, or the untreated area that was not ex- sure for the proton radiation induced activation of the coagulation posed to any cream other than the Tegaderm dressing. At 14 days cascade. Treatment with BeneFIX reduced the clotting times in ir- post-irradiation, biopsies were performed that revealed decreased radiated ferrets back to levels that were essentially equivalent to melanosomes, necrotic keratinocytes and melanin deposition in the those of the non-irradiated control ferrets areas of irradiated skin treated with mometasone compared to Since treatment with BeneFIX increases the concentration of Factor the untreated irradiated skin. Thus, it was concluded from these IX, a factor depleted post-irradiation, BeneFIX could have beneﬁcial studies that topical application of steroids mitigate skin toxicity effects on coagulation when administered after the radiation expo- produced by exposure to SPE-like proton radiation (Cengel, K.A. and Sanzari, J.K ., Unpublished data).
Phytonadione (vitamin K) is essential for post-translational modiﬁcation of a glutamate to a carboxylated-glutamate that isnecessary for Factor II, VII, IX, and X It 5.14. Transparent ﬁlm dressing for protection against proton therapy was also evaluated as a countermeasure for activation of the pro- induced skin reactions in humans ton radiation induced coagulation cascade. The results indicatethat phytonadione had a minor beneﬁcial effect on PT values in Patients undergoing proton therapy for prostate cancer fre- the proton irradiated ferrets, but did not affect the aPTT values quently develop radiation dermatitis. It has been reported that two prostate cancer patients undergoing proton cancer therapy at the Treatment of DIC with blood clotting factors or other compo- University of Pennsylvania had radiation dermatitis that appears nents of plasma is a topic that has been reviewed previously to have been substantially diminished by the presence of trans- We have demonstrated that SPE-like proton radia- parent ﬁlm dressings (Beekley stickers) In tion led to hypocoagulability by activating the clotting cascade that these studies, small circular (2.5 cm diameter) transparent adhe- consumes factors involved in coagulation. SPE-like proton radia- sive markers were placed on their skin to assist with daily align- tion can also cause leucopenia and severe lymphocytopenia, which, ment in these patients treated with a total proton dose of 79.2 Gy in combination with the effects of the SPE-proton radiation on in 1.8 Gy fractions, using two opposed lateral beams daily. It was hemostasis, could have major health consequences in irradiated observed that the covered areas of the skin exhibited considerably subjects. Our studies have shown that BeneFIX can serve as a po- diminished radiation dermatitis compared to the uncovered areas tential countermeasure for the increased bleeding times in ferrets of the skin, and this difference persisted for at least one month caused by exposure to SPE-like proton radiation.
after the therapy period ended. A phantom dosimetric study wasperformed to evaluate the impact of the transparent ﬁlm dressing 5.12. Corticosteroid as a countermeasure for radiation induced on a beam's SOBP, and the results indicated no gross dosimetric ef- pneumonitis and pneumonopathy in pigs fect. Thus, the transparent adhesive markers appear to have atten-uated radiation dermatitis in these two patients without affecting Several pigs exposed to simulated SPE radiation developed the SOBP. It is hoped that this ﬁnding can improve proton-related symptomatic, radiation-associated pneumonopathy that radio- radiation dermatitis in other types of treatments for cancer as well graphically involved all lung ﬁelds but was worse in the pleura and (i.e., in other conditions in which proton radiation exposure could apices where the radiation dose was found to be highest lead to radiation dermatitis).
A.R. Kennedy / Life Sciences in Space Research 1 (2014) 10–43 in ferrets exposed to SPE protons and γ -radiation and on peripheral hematopoietic cell (WBC, lymphocyte In recent years, we have been engaged in in vitro and ani- and neutrophil) counts in minipigs exposed to simulated proton mal studies on acute biological effects of the types of radiation at SPE or simulated electron SPE radiation (6 + 12 MeV electrons), the energies, doses and dose-rates relevant to space travel. Three which resulted in a comparable dose distribution to the proton species of animals, i.e., ferrets, pigs and mice, were used in our SPE radiation expected in an SPE, especially at the low end of studies to evaluate the effects of radiation on various biological the radiation dose range evaluated The re- endpoints including survival, cancer development, hematopoietic sults of other studies described here suggest that the effects of cell counts, emesis, blood coagulation, CNS endpoints (which in- proton radiation were comparable to those of the reference ra- cluded social exploration, submaximal exercise treadmill, and lo- diations evaluated. These studies include studies on the lack of comotor activity), cataract development, oxidative stress, gastroin- bacterial containment in the GI tract from exposure to γ -rays testinal tract bacterial translocation, immune activation, and gene or SPE proton radiation in mice on fatigue in expression associated with programmed cell death and ECM re- mice after irradiation with γ -rays or protons on the skin changes resulting from exposure to SPE For 30-day survival, 1-GeV/n proton radiation appeared to be proton or electron radiation on the develop- more lethal than X-rays to mice but ment of DIC in irradiated ferrets and on SPE-like proton radiation was comparable to γ -rays for ferrets mortality of mice exposed to radiation alone or radiation with Ferrets are considerably more sensitive than exposure to microgravity, along with a bacterial challenge (Drew mice to the lethal effects of radiation exposure. In experiments Weissman and colleagues, Unpublished data). Overall, the differ- with mice irradiated at lower doses of protons (3 Gy) or HZE parti- ences in the effects of the proton and reference radiation were cles (0.5 Gy), acute effects of the radiation doses were not observed relatively small or limited to the lower end of the dose range over a 30 day experimental period, but the long-term survival in (∼ 0.5 Gy) evaluated.
the irradiated mice was reduced signiﬁcantly by the radiation ex- The mechanism(s) for mortality in irradiated mammals is not posure, and the decreased long-term survival was accompanied by well understood, although numerous hypotheses have been pro- a signiﬁcant increase in the rate of development of malignant lym- posed. At the lowest total body radiation doses leading to mor- phomas and Harderian gland tumors, as well as the fractions of tality, death occurs from the hematopoietic syndrome, which is animals with malignant lymphoma or rare tumors believed to result from the cell killing effects of radiation in the bone marrow that lead to low numbers of circulating blood cells With respect to peripheral WBC and lymphocyte counts, the ef- and the resultant hematopoietic symptoms, such as infection and fects of proton irradiation given as a homogeneous dose to the bleeding due to the loss of leukocytes and platelets. Over the last mice is not affected by the dose fractionation, dose-rates or proton half century, the radiation dose required to kill half of the irradi- energy in the ranges evaluated. In contrast, simulated hypogravity ated subjects, known as the LD50, has been used as a parameter brought about by PWS was shown to potentiate splenic lympho- of radiation sensitivity for comparisons among various mammalian cytes to the cell killing effects of radiation species. It is well-known that the LD50 value is highly variable A number of other studies indicated that SPE radiation and sim- for different mammalian species; however, the bone marrow cell ulated microgravity produced by HS led to synergistic adverse ef- sensitivity to ionizing radiation is remarkably similar among dif- fects on hematopoietic and immune cell functions, including bacte- ferent species, strains and individuals rial containment in the GI tract T-cell activation These results suggest that the lethal ef- and death from a bacterial challenge fects of radiation on bone marrow and hematopoietic cells may not at a sub-lethal dose be the primary mechanism for radiation exposure related death.
For emesis in ferrets, the risk of SPE radiation-induced vom- Our studies in ferrets irradiated with SPE-like protons have sug- iting is low and may reach statistical signiﬁcance only when the gested that radiation induced activation of the coagulation cascade radiation dose reaches 1 Gy or higher. The ED10 and ED50 values may result in DIC, which could be a major mechanism by which estimated for the fraction of animals that retched or vomited af- relatively low doses of radiation lead to mortality ter proton irradiation at the high dose rate (0.5 Gy/minute) were The human LD50 values for radiation induced death lower than the lower limits of the respective 95% conﬁdence inter- are imprecise because there have been relatively few cases in vals established for γ -rays, suggesting that high dose rate proton which human subjects were exposed to radiation at doses near irradiation was more effective than high dose rate γ -ray irradia- the LD50, especially in the last half century. The estimated human tion in inducing retching and vomiting. There was a large sparing LD50 value for ionizing radiation ranges from 3 to 4 Gy for young effect observed at the low dose rates expected for SPE radiation, adults, without medical intervention to 2 to 3 Gy for the very such that the results for vomiting and retching in response to low young or the old Remarkably different dose-rate exposure to SPE radiation were not statistically signif- LD50 values have been reported for different species icant when compared to control animals. The trend, however, in the experiments performed at the low dose rate appeared to indi- as the most sensitive mammalian species cate more retching and vomiting episodes in ferrets irradiated with closely followed by dogs and pigs protons than in the animals exposed to γ -rays at the same dose The LD50 in Gottingen pigs is as low as 1.8 Gy and widespread hemorrhaging is observed at death of the irradiated The results from some of the studies described here indicated Gottingen pigs with some evidence of DIC that proton radiation had considerably more severe adverse effects at doses near the LD50, such as the rapid onset of systemic in- compared to those produced by comparable doses of the reference ﬂammation (C-reactive protein, ﬁbrinogen) and multi-organ dys- radiations used, which include x-rays, γ -rays and electrons. These function Similarly, dogs exhibit hemor- include studies on mouse survival after irradiation with protons rhagic diathesis at doses near the LD50, and die with signs re- and x-rays on ferret hematopoietic sembling DIC While DIC cell counts after exposure to SPE protons and γ -rays has not been diagnosed as a cause of radiation induced death on ferret retching and vomiting induced by SPE pro- in the pig or dog studies or in irradiated human populations, a tons and γ -rays on blood clotting times hallmark of DIC, i.e. hemorrhage, at death has been frequently A.R. Kennedy / Life Sciences in Space Research 1 (2014) 10–43 observed in irradiated mammals, including humans. There is ex- for the mammalian species. This hypothesis provides a novel and tensive evidence that widespread hemorrhages occurred in the reasonable explanation for the great variability observed in LD50 Hiroshima and Nagasaki atomic bomb casualties, even in the rel- values among different mammalian species.
atively low radiation dose groups with the There is a considerable amount of evidence that humans ex- estimated LD50 values of approximately 2.5 hibit hemorrhaging at doses near the LD50. The LD50 of the atom Other information about hemorrhaging in hu- bomb casualties has been estimated to be approximately 2.5 Gy mans after irradiation comes from accidental exposures in Norway The percent of those dying from exposure to the atom bombs with evidence of hemorrhage in which several people were accidentally exposed can be estimated from the atom bomb casualty data, which has to whole body irradiation at doses near the human LD50 and been reviewed by The casualty dose esti- widespread hemorrhages were observed.
mates are given in a report by the U.S. Atomic Energy Commission A reduction in the number of platelets can result in hemorrhag- As described by people ex- ing and death; however, our studies suggest that the blood clotting posed to radiation from the atom bomb used in Hiroshima have abnormalities in irradiated ferrets is not caused by a reduction been classiﬁed into several groups. In Group II (patients dying dur- in the number of platelets ing the third, fourth, ﬁfth and sixth weeks or surviving with severe A similar phenomenon has been reported clinical symptoms), some of the people lived and some of the peo- in irradiated dogs in which the platelet counts are not depressed ple died. It is expected that the people who died in Group II were in dogs at a "preterminal" stage in which bleeding abnormalities those who received a dose of radiation near the human LD50 level.
are observed, although the platelet counts are decreased greatly The data that were collected from each person represented one tis- in irradiated dogs with full-blown DIC In sue section from each tissue/organ examined, and the fraction of atom bomb casualties, widespread hemorrhages were observed in tissues exhibiting hemorrhage is reported by people before the level of platelet counts had fallen to levels ex- For many of the organs/tissues (e.g., kidney, liver and other or- pected to cause hemorrhage Furthermore, gans), the percentages exhibiting hemorrhage are as high as 60%, while platelet injections and other blood clotting factors can have but it is not clear what fraction of exposed individuals experienced beneﬁcial effects on hemorrhaging, platelet infusions do not pre- hemorrhaging in one or more organs. (For example, it is not indi- vent all deaths from the radiation exposure cated whether 60% of the livers exhibiting hemorrhage are from the same people as 60% of the kidneys exhibiting hemorrhage).
Hemorrhaging and signs of DIC have been frequently reported Therefore, it is assumed that 60% of the people exhibiting hemor- in higher mammalian species, such as dogs and pigs; however, rhage is a low estimate and that the true value lies between 60% hemorrhaging has not been reported in mice at doses near the and 100% of the people dying in Hiroshima (after exposure at a 50. Mice are the animals that are most often used in radiobi- 50) that had evidence of hemorrhage. These data ology studies. Bacteremia is the leading cause of death in mice indicate that the atom bomb casualties are likely to have had very high frequencies of hemorrhaging at death, similar to those ob- orrhage is thought to be the major cause of death in dogs, rab- served for other large mammals (e.g., pigs and dogs).
bits, guinea pigs or pigs It is also noteworthy that knowledge obtained about the ad- after irradiation at doses near the LD verse health effects of inhomogeneous doses of interventional and species. The pathophysiology of the hematopoietic syndrome in therapeutic radiation is relevant for evaluation of radiation ef- pigs is thought to be similar to that observed in humans fects in people resulting from exposure to interventional radiology Such differences suggest that we need new ways procedures or therapeutic radiation, in which the skin doses are of thinking about mechanisms for death after irradiation at doses higher than the internal organ doses. An example of the rele- vance for patients on earth is that high doses of SPE-like radiation 50 level. Since the human LD50 is closer to the LD50 of ferrets, dogs and pigs than to the LD primarily to the skin with minimal to no signiﬁcant doses to in- 50 of mice, species such as fer- rets, dogs and pigs are likely to be better animal models than mice ternal organs have been shown to produce adverse health effects for evaluating the effects of radiation and radiation countermea- in internal organs (e.g., pulmonary toxicities or pneumonitis withcoughing in the lungs, bone marrow changes and reductions in cir- sures on bleeding, coagulation cascade activation and DIC risks for culating blood cell counts It is noteworthy that the LD50 values for these larger mammals 7. Summary concerning the major effects of SPE and space
are considerably lower than those observed in various strains of mice Examples of some published LD50values for mammals are as follows: Ferrets – 1.5 Gy 7.1. Dosimetry Gottingen pigs – 1.8 Gy pigs – av-erage value – 2.6 Gy dogs – average value With the incorporation of modern radiation oncology ap- – 2.6 Gy monkeys – 5.07 Gy proaches, such as CT based Monte Carlo dosimetry, into the Human mice – average value – 8.16 Gy Space Program, it has become feasible to accurately predict organ Using published data, we have compared the per- speciﬁc radiation doses for astronauts exposed to SPE radiation.
centage of animals exhibiting hemorrhage at death (from exposure Depending on the organ system of interest (deep vs. superﬁcial) to radiation doses near the LD50 level) as a function of the LD50 and the ﬂuence/energy proﬁle of the exposure (hard vs. soft event), of the species and observed an excellent correlation between the either the physical size of the astronaut or the ﬂuence/energy pro- percent of animals developing "DIC" (from hemorrhaging at death, ﬁle for the SPE can be the determining factor for radiation induced the hallmark of DIC) and the LD50 value for the species/strain (Krigsfeld, G.S. and Kennedy, A.R., Unpublished data). These ﬁg-ures for animal hemorrhaging at death range from 100% of ferrets 7.2. Adverse effects in hematopoietic/immune system cells – to 0% of the mice (as mice irradiated at radiation doses neartheir LD50 levels die from bacteremia Signiﬁcant decreases in WBC counts were observed in mice and Based on this analysis, it is hypothesized that ferrets irradiated with SPE-like proton and γ -ray radiation at to- the propensity to develop DIC has a role in determining the LD50 tal body doses of 0.5 to 2 Gy, and in pigs irradiated with proton A.R. Kennedy / Life Sciences in Space Research 1 (2014) 10–43 simulated SPE radiation at skin doses of 2 to 10 Gy and elec- 7.6. Disseminated intravascular coagulation tron simulated SPE radiation at skin doses of 2.5 to 25 Gy. At thehigher doses of proton or γ -ray radiation, the neutrophil counts in Both SPE-like proton and γ -ray radiation at doses near 2 Gy re- the blood of both mice and ferrets, but not pigs, reached critically sulted in DIC in ferrets, which led to 100% mortality. The threshold low levels that, if observed in a patient in a hospital (e.g., follow- dose of radiation for this effect is 1.5 Gy. This is an important ﬁnd- ing radiation or chemotherapy for cancer), would trigger a medical ing, as DIC has not been established previously as a cause of death response and suggest the use of countermeasures to increase the in mammals following radiation exposure. The blood clotting ab- level of neutrophils. In the pigs exposed to proton simulated SPE normalities in ferrets were observed at a very low dose (0.25 Gy, radiation, the neutrophil counts did not return to normal levels which was the lowest dose evaluated in the studies), and radia- even at months after the radiation exposure.
tion at the SPE-like low dose rate resulted in more severe effectson the blood clotting parameters in ferrets than the high dose-rate 7.3. Adverse effects on the immune system Yucatan minipigs exposed to a 2.5 Gy dose of radiation were also diagnosed with DIC and died (or were euthanized). Minipigs At a dose of 2 Gy, SPE-like proton radiation or γ -ray radiation exposed to a 2 Gy dose of SPE like proton radiation did not die, was shown to cause breaks in the epithelial layer of the small in- but they exhibited severe clotting abnormalities (increased bleed- testine of mice, which allows translocation of bacteria and bacterial ing times) that could be problematic during space travel.
products, such as LPS. The threshold for the morbidity/mortality Based on the work in ferrets and pigs performed as part of the effect in bacterial challenge studies was between 1.0 and 1.5 Gy.
space radiation studies described here, it is hypothesized that DIC The proton radiation works synergistically with HS to result in an may be a major cause of death in humans following exposure to accumulation of a relatively large amount of LPS in the intervil- relatively low doses of radiation.
lous regions of the ileum. Due to the synergistic effect from HS,the threshold doses for immune system parameters determined 7.7. Changes potentially related to the development of vision without the HS treatment may underestimate the actual immune system risks for astronauts during space travel involving micro-gravity conditions.
It was observed that pigs exposed to 2.5–7.5 Gy (skin dose) Another major adverse effect observed in immune system of simulated SPE – electron radiation (October 1972 event) exhib- cells is the lack of, or reduction in the level of, activation in T- ited increased opening pressure values, which lasted up to 90 days lymphocytes in mice exposed to 1–2 Gy of SPE-like proton or post-radiation. Other endpoints have also been evaluated which γ -ray radiation, either with or without simulated microgravity (us- could have signiﬁcance for the ﬁndings that many astronauts ex- ing the PWS and HS systems).
posed to long-duration space ﬂight have exhibited vision alter-ations. As one example, increased nerve sheath diameters (docu- 7.4. Emesis (vomiting and retching) mented by ocular ultrasound technology) have been observed inpigs exposed to SPE-like radiation. Increased nerve sheath diame- In female descented Fitch ferrets, irradiation with 60Co γ -rays ters have also been documented by ocular ultrasound technology or 155-MeV protons at a high dose rate of 0.5 Gy/minute resulted in astronauts and this ﬁnding has been implicated in the develop- in dose-dependent changes in the endpoints that are indicative of ment of astronaut vision alterations retching and vomiting. The minimum radiation doses required to A relatively low dose of SPE-like radiation (skin doses as low induce statistically signiﬁcant changes in retching- and vomiting- as 2.5 Gy) can lead to increased intracranial pressure in pigs, and related endpoints were 0.75 and 1.0 Gy, respectively. The RBE of a dose of 5 Gy of proton simulated SPE radiation was shown to the proton radiation at the high dose rate (relative to the γ -rays at increase the nerve sheath diameter. The statistically signiﬁcant ef- the same dose rate) did not differ signiﬁcantly from 1. Similar, but fects observed on these endpoints in the pig studies suggest that smaller and less consistent, changes in the retching- and vomiting- astronaut exposure to these relatively low doses of SPE radiation related endpoints were also observed for ferrets irradiated with could exacerbate the vision alterations known to exist in astro- γ -rays and protons at the low dose rate of 0.5 Gy/hour. Since this nauts during future exploration class missions.
low dose rate is similar to a radiation dose rate expected duringa SPE, these results suggest that the risk of SPE radiation-induced 7.8. Threshold radiation doses for statistically signiﬁcant adverse vomiting is low and is likely to reach statistical signiﬁcance only biological effects from SPE-like radiation in vivo when the radiation dose reaches 1 Gy or higher.
The estimated threshold radiation doses to cause signiﬁcant ad- verse biological effects in vivo for various endpoints measured are 7.5. Skin effects summarized as follows: At skin doses of 7.5 Gy and higher, there is a persistent im- • Reductions in WBC counts in mice and ferrets: a homogeneous munological dysfunction in pig skin, which is characterized by an dose of 0.5 Gy; in pigs: a homogeneous dose of 2.0 Gy (lowest enhanced DTH response; a similar effect was observed in mice dose evaluated) of SPE-like proton radiation.
at doses of 2 Gy or less. At higher doses of radiation, the pig • Levels of death in mice exposed to SPE like protons, along with skin becomes more sensitive to touch (lymphedematous), and HS, in response to a bacterial challenge: 1.0 to 1.5 Gy.
there can be blistering, burns and epithelial dysfunction (e.g., pig- • Elevated levels of LPS and LPB in mouse serum: 2 Gy of SPE- ment incontinence, which indicates defective cell to cell transfer like proton radiation.
of biomolecules). The threshold radiation doses for the skin effects • Increases in blood clotting time in ferrets: 0.25 Gy of SPE-like are 4.5 to 5 Gy. The decrease in vascular bed area is the most se- protons at a low, SPE-like dose rate.
rious skin effect potentially resulting from irradiation at very high • Lack of T cell activation in mice: 1 Gy.
skin doses. However, this complication would be a highly unlikely • Skin effects: skin hyperpigmentation, epidermal thickening, occurrence for astronauts since such high radiation doses are not and decrease in vascular bed area: 4.5 to 5 Gy of pSPE or eSPE expected during most SPEs.
A.R. Kennedy / Life Sciences in Space Research 1 (2014) 10–43 • Emesis in ferrets (for high dose-rate irradiation): 1 and 0.75 Gy 7.10. Dose-rate effects for vomiting and retching, respectively. For low SPE like dose-rates, the results were not statistically signiﬁcant at doses up One major endpoint evaluated that was affected by dose rate to 2 Gy for SPE like proton radiation compared to controls.
was ferret emesis, which was signiﬁcantly increased after irradia- • Levels of death from, or signs of, DIC in ferrets or pigs. All fer- tion with SPE-like proton radiation or γ -ray radiation administered rets died with signs of DIC at a dose of 2 Gy from SPE-like at the high dose rate of 0.5 Gy/minute, but not at a low dose rate protons or γ -ray radiation. The threshold dose for this effect of 0.5 Gy/hour. The other major endpoint affected by dose-rate is 1.5 Gy. Ferrets are thought to be more susceptible to radi- was the blood clotting time, which was increased to a considerably ation induced DIC than humans, while the sensitivity of pigs greater extent in the studies with the low, SPE-like dose-rate than to radiation induced DIC may be more like that of humans. In in the high dose-rate radiation studies. For all other endpoints, the an experiment in which 3 Yucatan minipigs were exposed to sparing effects of the radiation dose rate are either insigniﬁcant or X-rays at a dose of 2.5 Gy, one pig died and another pig was minimal and not biologically meaningful.
euthanized; both of these pigs had evidence of DIC. 2.5 Gy is 8. Agents identiﬁed as countermeasures for space radiation
a potentially lethal dose of radiation for humans as well. At induced adverse biological effects
an SPE-like radiation dose of 2.0 Gy, pigs had severe clottingabnormalities suggestive of DIC. With the other space stres- As a part of the work for this program, the following agents sors (e.g., microgravity, elevated levels of oxygen during EVAs have been identiﬁed as countermeasures, which may lead to risk and carbon dioxide in the spacecraft, etc.), the lethal human reductions in astronauts exposed to space radiations.
dose could be < 2.5 Gy and astronauts may be susceptible to A. Dietary antioxidants fructose the onset and progression of DIC at doses that could be re- and G-CSFs (Neupogen, Neulasta) ceived from exposure to SPE radiation. In any case, astronauts as countermeasures to prevent or alleviate the loss of circulating are likely to have bleeding abnormalities from very low SPE white blood cells radiation doses (25 cGy or below), and they should avoid ex- posure to anything that could damage the skin integrity due to the bleeding risk following a sizeable exposure to SPE radi- B. Orally administered antibiotics (e.g., enroﬂoxacin) as a coun- termeasure to prevent or alleviate translocation of bacteria andbacterial products as well as death from bacterial challenge in ani- 7.9. RBE values mals exposed to SPE-like proton radiation and hindlimb suspension(Drew Weissman, Unpublished data).
A higher RBE value for a given effect indicates that a more se- C. 5-HT3 antagonists (e.g., oral ondansetron [Zofran]) as a coun- vere effect is expected for exposure to SPE radiation than from termeasure for proton radiation induced emesis (retching and conventional reference radiations (e.g., γ -rays, x-rays or electrons).
In our studies, it was observed that there can be: 1) different RBEs D. Corticosteroid therapy as a countermeasure for pneumonopa- for different biological endpoints in the same animal species/strain, thy/pneumonitis that developed after radiation exposure and 2) different RBEs for the same biological endpoint in differ- E. Topically applied steroid cream (mometasone-Elecon) (Cen- ent species/strains. However, for most of the endpoints evaluated, gel, K.A. and Sanzari, J.K., Unpublished data) and transparent ﬁlm which include hematopoietic blood cell counts, immune system dressing as countermeasures for radiation in- parameters and fatigue measured in mice, emesis evaluated in duced skin damage (e.g. hyperpigmentation).
ferrets, skin effects assessed in pigs, the RBE values were not sig- F. Dietary antioxidants as a countermeasure to decrease the risk niﬁcantly different from 1 except for the following: of long-term radiation effects, e.g. cancer Hematopoietic blood cell counts in ferrets: RBE ranges from G. Beneﬁx as a countermeasure for increased bleeding times 1.2 to 1.6 for the WBC count at 48 hours after irradiation, RBE after radiation exposure ranges from 1.9 to 2.1 for neutrophil count at 48 hours afterirradiation (RBE values were particularly increased at the low end of the SPE proton dose range evaluated); • Hematopoietic blood cell counts in pigs: RBE ranges from 2.4 Most of the research investigations from the author's labo- to 4.1 for the WBC count, and 2.2 to 5.0 for the neutrophil ratory discussed in this review were supported by the National count on day-4 post-irradiation (RBE values were particularly Space Biomedical Research Institute (NSBRI). The NSBRI is funded increased at the low end of the SPE proton dose range evalu- through NASA NCC 9-58. In addition, some studies were funded by a NASA grant (NAG9-1517). Several trainees participating in theseinvestigations were supported by the NIH-funded Radiation Biol- RBE values were determined for hematopoietic cell counts in ogy Training Grant (T32CA009677).
mice, ferrets and pigs; therefore, they can be compared acrossthese three species. From these data, it was observed that the RBE values of the SPE-like radiation for white blood cell counts varygreatly between mice, ferrets and pigs, with the RBE values being somewhat greater in ferrets than in mice, and considerably greater in pigs than in ferrets or mice. This trend suggests that the RBE values of SPE radiation for white blood cell counts could be con- siderably greater in humans than those observed in smaller mam- mals, and that SPE proton radiation may be far more hazardous to humans than previously estimated from studies performed in small animals (e.g., rodents).
A.R. Kennedy / Life Sciences in Space Research 1 (2014) 10–43 Czarnik, T.R., 1988. Medical Emergencies in Space.
A.R. Kennedy / Life Sciences in Space Research 1 (2014) 10–43 Hlatky, L., Hahnfeldt, P., 2014. Beyond the cancer cell: progression-level determi- nants highlight the multiscale nature of carcinogenesis risk. Cancer Res. 74 (3), 659–664. Epub 2013 Nov 22.
PMID: 24272486.
A.R. Kennedy / Life Sciences in Space Research 1 (2014) 10–43 Krigsfeld, G.S., Savage, A.R., Billings, P.C., Lin, L., Kennedy, A.R., 2014. Evi- dence for radiation induced disseminated intravascular coagulation as a major cause of radiation induced death in ferrets. Int. J. Radiat. Oncol. Biol. Phys.
pii S0360-3016(13)03611-0 [Epub ahead of print].
A.R. Kennedy / Life Sciences in Space Research 1 (2014) 10–43 NCRP, 1989. National Council on Radiation Protection and Measurements (NCRP) Report No. 98. Guidance on radiation received in space activities. NCRP.
NCRP, 2006. National Council on Radiation Protection and Measurements (NCRP) Report No. 153. Information needed to make radiation protection recommenda- tions for space missions beyond low-earth orbit. NCRP.
NCRP, 2010. National Council on Radiation Protection and Measurements (NCRP) Report No. 168. Radiation dose management for ﬂuoroscopically-guided inter- ventional medical procedures. NCRP.
NCRP, 2011. National Council on Radiation Protection and Measurements (NCRP) Re- port No. 170: Second primary cancers and cardiovascular disease after radiation therapy. NCRP.
A.R. Kennedy / Life Sciences in Space Research 1 (2014) 10–43 Romero-Weaver, A.L., Ni, J., Lin, L., Kennedy, A.R., 2014. Orally administered fruc- tose increases the numbers of peripheral lymphocytes reduced by expo- sure of mice to gamma or SPE-like proton radiation. Life Sci. Space Res.
Sanzari, J.K., Wan, X.S., Rusek, A., Diffenderfer, E.S., Kennedy, A.R., 2014. Acute hematological effects in mice exposed to the expected doses, dose-rates, and energies of solar particle event-like proton radiation. Life Sci. Space Res.
A.R. Kennedy / Life Sciences in Space Research 1 (2014) 10–43

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Available Online through www.ijpbs.com (or) www.ijpbsonline.com IJPBS Volume 3 Issue 4 OCT-DEC 2013 255-264 Research Article Pharmaceutical Sciences METHOD DEVELOPMENT AND VALIDATION OF RP-HPLC METHOD FOR SIMULTANEOUS ESTIMATION OF DICYCLOMINE HYDROCHLORIDE AND DICLOFENAC POTASSIUM IN TABLET DOSAGE FORMS

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Diseases and ConditionsUlcerative colitis By Mayo Clinic Staff Ulcerative colitis (UL-sur-uh-tiv koe-LIE-tis) is an inflammatory bowel disease (IBD) thatcauses long-lasting inflammation and ulcers (sores) in your digestive tract. Ulcerativecolitis affects the innermost lining of your large intestine (colon) and rectum. Symptomsusually develop over time, rather than suddenly.