Comparative study of spermatogonial survival after x-ray exposure, high LET (HZE) irradiation or spaceflight.

Spermatogonial cell loss has been observed in rats flown on Space Lab 3, Cosmos 1887, Cosmos 2044 and in mice following irradiation with X-ray or with high energy (HZE) particle beams. Spermatogonial loss is determined by cell counting in maturation stage 6 seminiferous [correction of seminferous] tubules. With the exception of Iron, laboratory irradiation experiments (with mice) revealed a similar pattern of spermatogonial loss proportional to the radiation dose at levels less than 0.1 Gy. Helium and Argon irradiation resulted in a 5% loss of spermatogonia after only 0.01 Gy exposure. However, significant spermatogonial loss (45%) occured at this radiation level with Iron particle beams. The loss of spermatogonia during each space flight was less than 10% when compared to control (non-flight) animals. This loss, although small, was significant. Although radiation may be a contributing factor in the loss of spermatogonia during space flight, exposure levels, as determined by dosimetry, were not significant to account for the total cell loss observed.     

Protective effects of melatonin against C beam irradiation-induced oxidative stress and DNA injury in the mouse brain

The purpose of this experiment was to estimate the protective effects of melatonin against radiation-induced brain damages in mice induced by heavy ion beams. Kun-Ming mice were randomly divided into five groups: normal control group, irradiation control group, and three different doses of melatonin (5, 10, and 20 mg/kg, i.p.) treated groups. Apart from the normal control group, the other four groups were exposed to whole-body 4.0 Gy carbon ion beam irradiation (approximately 0.5 Gy/min) after i.p. administration of normal saline or melatonin 1 h before irradiation. The oxidative redox status of brain tissue was assessed by measurement of malondiadehyde (MDA) levels, total superoxide dismutase (T-SOD), cytosolic superoxide dismutase (Cu/ZnSOD, SOD1) and mitochondrial superoxide dismutase (MnSOD, SOD2) activities at 8 h after irradiation. DNA damages were determined using the Comet assay and apoptosis and cell cycle distribution were detected by flow cytometric analyses. A dramatic dose-dependent decrease in MDA levels, tail moment, rates of tailing cells, and apoptosis, and a dose-dependent increase in T-SOD and SOD2 activities, in brain tissues in the melatonin-treated groups were detected compared with the irradiation only group. Furthermore, flow cytometric analysis demonstrated that the percentage of brain cells in the G0/G1 phase decreased significantly, while those in the S and G2/M stage increased dramatically, with mice pretreated with melatonin compared to the irradiation control group. These data indicate that melatonin has protective effects against irradiation-induced brain injury, and that its underlying protective mechanisms may relate to modulation of oxidative stress induced by heavy ionirradiation.     

A low dose pre-irradiation induces radio- and heat-resistance via HDM2 and NO radicals,and is associated with p53 functioning

The aim of this work was to clarify the effect of low dose pre-irradiation on radio- and heat-sensitivity. Wild-type (wt) p53 and mutated (m) p53 cells derived from the human lung cancer H1299 cell line were used. The parental H1299 cell line is p53-null. Cellular sensitivities were determined with a colony-forming assay. When wtp53 cells were exposed to a low dose X-irradiation, induction of radio- and heat-resistance was observed only in the absence of RITA (an inhibitor of p53-HDM2 interactions), aminoguanidine (an iNOS inhibitor) and c-PTIO (an NO radical scavenger). In contrast, the induced radio- and heat-resistance was not observed under similar conditions in mp53 cells. Moreover, heat-resistance as well as radio-resistance developed when wtp53 cells were treated with ISDN (an NO generating agent) alone. These findings suggest that NO radicals are an initiator of radio- and heat-resistance, and function through the activation of HDM2 and the depression of p53 accumulation.     

Cellular responses by exposure to heavy-ions.

To better understand cellular responses in human lymphoblastoid cell TK6 after exposure to C-ion (22 keV/micrometer) and Fe-ion (1000 keV/micrometer), both protein induction and cell-cycle progression have been extensively analyzed by the recently developed techniques. While proceeding this line of analyses, we realized the importance of studying low-dose effect, in relation to the genetic alterations. Adaptive response by 5~20 cGy of such C- or Fe-ion irradiation to both lethal and mutagenic effects of the challenging X-ray exposure (1~3 Gy) was difficult to be seen in this TK6 cells, but surprisingly, a relatively high level of p53 and its related proteins induction was observed after low-dose irradiations of heavy-ions. Here, we focus to introduce the above results of genetic and biochemical studies to elucidate the adaptive response.     

Analysis of cytogenetic effects of the secondary radiation resulting from 70 GeV protons of Chinese hamster cells.

The cell culture of a Chinese hamster was irradiated on a Serpuchov proton synchrotron at a dose of 0.5-4 Gy and a dose rate of 1 Gy/min and by gamma-irradiation at dose 1-5 Gy and dose rate 1.2-1.4 Gy/min. The effect of radiation on the cell culture was judged from chromosomal aberrations in G2-stage of cell cycle and micronuclear test. The relative biological efficience of the secondary radiation was approximately 3. Modifying effect of caffeine on the cells irradiated by secondary radiation of synchrotron was not observed. In the presence of caffeine the effect of gamma-irradiation practically is increased up to the level observed upon secondary irradiation. This suggests that secondary radiation inhibits the repair of the cytogenetic damage.     

The effects of proton exposure on neurochemistry and behavior.

Future space missions will involve long-term travel beyond the magnetic field of the Earth, where astronauts will be exposed to radiation hazards such as those that arise from galactic cosmic rays. Galactic cosmic rays are composed of protons, alpha particles, and particles of high energy and charge (HZE particles). Research by our group has shown that exposure to HZE particles, primarily 600 MeV/n and 1 GeV/n 56Fe, can produce significant alterations in brain neurochemistry and behavior. However, given that protons can make up a significant portion of the radiation spectrum, it is important to study their effects on neural functioning and on related performance. Therefore, these studies examined the effects of exposure to proton irradiation on neurochemical and behavioral endpoints, including dopaminergic functioning, amphetamine-induced conditioned taste aversion learning, and spatial learning and memory as measured by the Morris water maze. Male Sprague-Dawley rats received a dose of 0, 1.5, 3.0 or 4.0 Gy of 250 MeV protons at Loma Linda University and were tested in the different behavioral tests at various times following exposure. Results showed that there was no effect of proton irradiation at any dose on any of the endpoints measured. Therefore, there is a contrast between the insignificant effects of high dose proton exposure and the dramatic effectiveness of low dose (<0.1 Gy) exposures to 56Fe particles on both neurochemical and behavioral endpoints.     

Induction of genomic instability after an acute whole-body exposure of mice to Fe ions

The purpose of this study was to evaluate dose–response relationships for the in vivo induction of micronuclei (MN) as a measure of both initial radiation damage and the induction of genomic instability. These measurements were made in mouse blood erythrocytes as a function of radiation dose, radiation quality, time after irradiation, and the genetic background of exposed individuals. Blood samples were collected from two strains of mouse (CBA/CaJ and C57BL/6J) at different times up to 3 months following a whole-body exposure to various doses of 1 GeV/amu ⁵⁶Fe ions (0, 0.1, 0.5 and 1.0 Gy, at the dose rate of a 1 Gy/min) or ¹³⁷Cs gamma rays (0, 0.5, 1.0 and 3.0 Gy, at the dose rate of 0.72 Gy/min). Blood-smear slides were stained with acridine orange (AO). The frequencies of MN were measured in mature normochromatic-erythrocytes (MN-NCEs) and in immature polychromatic-erythrocytes (MN-PCEs). Effects of both types of radiation on erythropoiesis were also evaluated. As a measure of cell progression delay, a dose-dependent decrease in numbers of PCEs was observed at day 2 post-exposure in both strains, regardless of radiation quality. Subsequently, the levels of PCEs increased in all exposed mice, reaching control levels (or higher) by day 7 post-exposure. Further, at day 2 after the exposure, there was no increase in the frequency of MN-PCEs in CBA/CaJ mice exposed to ⁵⁶Fe ions while the frequency of MN-PCEs elevated as a function of dose in the C57BL/6J mice. At day 4, there was no dose related increase in MN-NCEs in either strain of mouse exposed to ¹³⁷Cs gamma rays. Additionally, at the early sacrifice times (days 2 and 4), ⁵⁶Fe ions were slightly more effective (per unit dose) in inducing MN-NCEs than ¹³⁷Cs gamma rays in CBA/CaJ mice. However, there was no increase in the frequency of MN-NCEs at late times after an acute exposure to either type of radiation. In contrast, both types of radiation induced increased MN-PCEs frequencies in irradiated CBA/CaJ mice, but not C57BL/6J mice, at late times post-exposure. This finding indicates the potential induction of genomic instability in hematopoietic cells of CBA/CaJ mice by both types of radiation. The finding also demonstrates the influence of genetic background on radiation-induced genomic instability in vivo.     

Head and neck tumors after energetic proton irradiation in rats.

This is a two-year progress report on a life span dose-response study of brain tumor risk at moderate to high doses of energetic protons. It was initiated because a joint NASA/USAF life span study of rhesus monkeys that were irradiated with 55-MeV protons (average surface dose, 3.5 Gy) indicated that the incidence of brain tumors per unit surface absorbed dose was over 19 times that of the human tinea capitis patients whose heads were exposed to 100 kv x-rays. Examination of those rats that died in the two-year interval after irradiation of the head revealed a linear dose-response for total head and neck tumor incidence in the dose range of 0-8.5 Gy. The exposed rats had a greater incidence of pituitary chromophobe adenomas, epithelial and mesothelial cell tumors than the unexposed controls but the excessive occurrence of malignant gliomas that was observed in the monkeys was absent in the rats. The estimated dose required to double the number of all types of head and neck tumors was 5.2 Gy. The highest dose, 18 Gy, resulted in high mortality due to obstructive squamous metaplasia at less than 50 weeks, prompting a new study of the relative biological effectiveness of high energy protons in producing this lesion.     

Spaceflight-relevant types of ionizing radiation and cortical bone: Potential LET effect?

Extended exposure to microgravity conditions results in significant bone loss. Coupled with radiation exposure, this phenomenon may place astronauts at a greater risk for mission-critical fractures. In a previous study, we identified a profound and prolonged loss of trabecular bone (29–39%) in mice following exposure to an acute, 2 Gy dose of radiation simulating both solar and cosmic sources. However, because skeletal strength depends on trabecular and cortical bone, accurate assessment of strength requires analysis of both bone compartments. The objective of the present study was to examine various properties of cortical bone in mice following exposure to multiple types of spaceflight-relevant radiation. Nine-week old, female C57BL/6 mice were sacrificed 110 days after exposure to a single, whole body, 2 Gy dose of gamma, proton, carbon, or iron radiation. Femora were evaluated with biomechanical testing, microcomputed tomography, quantitative histomorphometry, percent mineral content, and micro-hardness analysis. Compared to non-irradiated controls, there were significant differences compared to carbon or iron radiation for only fracture force, medullary area and mineral content. A greater differential effect based on linear energy transfer (LET) level may be present: high-LET (carbon or iron) particle irradiation was associated with a decline in structural properties (maximum force, fracture force, medullary area, and cortical porosity) and mineral composition compared to low-LET radiation (gamma and proton). Bone loss following irradiation appears to be largely specific to trabecular bone and may indicate unique biological microenvironments and microdosimetry conditions. However, the limited time points examined and non-haversian skeletal structure of the mice employed highlight the need for further investigation.     

Effects of carbon ions on primary cultures of mouse brain cells.

Primary mixed cultures of astrocytes and microglia were obtained from neonatal mice, and were irradiated with high-LET carbon ions. Immunohistochemical staining showed astrocytes survived more prominently than microglia. Tagged with specific antibodies, astrocytes and microglia surviving after irradiation were counted by flow cytometry. Decreases in the number of microglia and astrocytes were detected at a dose as small as 2 Gy when Day 5 cultures were irradiated with 13 keV/micrometer carbon ions. When the cultures were irradiated on Day 10, the dose-dependent decrease of microglia was more prominent for 13 keV/micrometer carbon ions than 70 keV/micrometer carbon ions. Astrocytes showed a marginal decrease at Day 10 and Day 14. We concluded that microglia are more sensitive than astrocytes to carbon ions and X-rays, and that the radiosensitivity of microglia depends on both differentiation/proliferation status and radiation quality.     

Low-dose carbon ion irradiation effects on DNA damage and oxidative stress in the mouse testis

To investigate the effects of low-dose carbon ion irradiation on reproductive system of mice, the testes of outbred Kunming strain mice were whole-body irradiated with 0, 0.05, 0.1, 0.5 and 1 Gy, respectively. We measured DNA double-strand breaks (DNA DSBs) and oxidative stress parameters including malondialdehyde (MDA) content, superoxide dismutase (SOD) activity, and testis weight and sperm count at 12 h, 21 d and 35 d after irradiation in mouse testis. At 12 h postirradiation, a significant increase in DNA DSB level but no pronounced alterations in MDA content or SOD activity were observed in 0.5 and 1 Gy groups compared with the control group. At 21 d postirradiation, there was a significant reduction in sperm count and distinct enhancements of DSB level and MDA content in 0.5 and 1 Gy groups in comparison with control. At 35 d postirradiation, the levels of DNA DSBs and MDA, and SOD activity returned to the baseline except for the MDA content in 1 Gy (P < 0.05), while extreme falls of sperm count were still observed in 0.5 (P < 0.01) and 1 Gy (P < 0.01) groups. For the 0.05 or 0.1 Gy group, no differences were found in DNA DSB level and MDA content between control and at 12 h, 21 d and 35 d after irradiation, indicating that lower doses of carbon ion irradiation have no significant influence on spermatogenesis processes. In this study, male germ cells irradiated with over 0.5 Gy of carbon ions are difficult to repair completely marked by the sperm count. Furthermore, these data suggest that the deleterious effects may be chronic or delayed in reproductive system after whole-body exposure to acute high-dose carbon ions.     

Effects of 40Ar and 56Fe ions on retinal photoreceptor cells of the rabbit: implications for manned missions to Mars.

Losses of photoreceptor cells (rods) from the retinas of New Zealand white (NZW) rabbits were detectable within 2 years after localized acute irradiation of optic and proximal tissues with > or = 7 Gy of 530 MeV u-1 40Ar ions or > or = 2 Gy of 465 MeV u-1 56Fe ions in the Bragg plateau region of energy deposition. Those limits were determined only from an analysis of variance of dose groups because the shapes of the dose response curves at early post-irradiation times are not known, a concern being addressed by experiments in progress. Losses of photoreceptor cells for the period 0.5-2.5 years post-irradiation, determined by provisional linear regression analysis, were approximately 1.7% Gy-1 and 2.5% Gy-1 for 40Ar and 56Fe ions, respectively.     

Radioprotection by metals: selenium.

The need exists for compounds that will protect individuals from high-dose acute radiation exposure in space and the agents that might be less protective but less toxic and longer acting. Metals and metal derivatives provide a small degree of radioprotection (dose reduction factor < or = 1.2 for animal survival after whole-body irradiation). Emphasis is placed here on the radioprotective potential of selenium (Se). Both the inorganic salt, sodium selenite, and the organic Se compound, selenomethionine, enhance the survival of irradiated mice (60Co, 0.2 Gy/min) when injected IP either before (-24 hr and -1 hr) or shortly after (+15 min) radiation exposure. When administered at equitoxic doses (one-fourth LD10; selenomethionine = 4.0 mg/kg Se, sodium selenite = 0.8 mg/kg Se), both drugs enhanced the 30-day survival of mice irradiated at 9 Gy. Survival after 10-Gy exposure was significantly increased only after selenomethionine treatment. An advantage of selenomethionine is lower lethal and behavioral toxicity (locomotor activity depression) compared to sodium selenite, when they are administered at equivalent doses of Se. Sodium selenite administered in combination with WR-2721, S-2-(3-aminopropylamino)ethylphosphorothioic acid, enhances the radioprotective effect and reduces the lethal toxicity, but not the behavioral toxicity, of WR-2721. Other studies on radioprotection and protection against chemical carcinogens by different forms of Se are reviewed. As additional animal data and results from human chemoprevention trials become available, consideration also can be given to prolonged administration of Se compounds for protection against long-term radiation effects in space.     

Tissue responses to low protracted doses of high LET radiations or photons: early and late damage relevant to radio-protective countermeasures.

Early and late murine tissue responses to single or fractionated low doses of heavy charged particles, fission-spectrum neutrons or gamma rays are considered. Damage to the hematopoietic system is emphasized, but results on acute lethality, host response to challenge with transplanted leukemia cells and life-shortening are presented. Low dose rates per fraction were used in some neutron experiments. Split-dose lethality studies (LD 50/30) with fission neutrons indicated greater accumulation of injury during a 9 fraction course (over 17 days) than was the case for gamma-radiation. When total doses of 96 or 247 cGy of neutrons or gamma rays were given as a single dose or in 9 fractions, a significant sparing effect on femur CFU-S depression was observed for both radiation qualities during the first 11 days, but there was not an earlier return to normal with dose fractionation. During the 9 fraction sequence, a significant sparing effect of low dose rate on CFU-S depression was observed in both neutron and gamma-irradiated mice. CFU-S content at the end of the fractionation sequence did not correlate with measured LD 50/30. Sustained depression of femur and spleen CFU-S and a significant thrombocytopenia were observed when a total neutron dose of 240 cGy was given in 72 fractions over 24 weeks at low dose rates. The temporal aspects of CFU-S repopulation were different after a single versus fractionated neutron doses. The sustained reduction in the size of the CFU-S population was accompanied by an increase in the fraction in DNA synthesis. The proliferation characteristics and effects of age were different for radial CFU-S population closely associated with bone, compared with the axial population that can be readily aspirated from the femur. In aged irradiated animals, the CFU-S proliferation/redistribution response to typhoid vaccine showed both an age and radiation effect. After high single doses of neutrons or gamma rays, a significant age- and radiation-related deficiency in host defense mechanisms was detected by a shorter mean survival time following challenge with transplantable leukemia cells. Comparison of dose-response curves for life shortening after irradiation with fission-spectrum neutrons or high energy silicon particles indicated high initial slopes for both radiation qualities at low doses, but for higher doses of silicon, the effect per Gy decreased to a value similar to that for gamma rays. The two component life-shortening curve for silicon particles has implications for the potential efficacy of radioprotectants. Recent studies on protection against early and late effects by aminothiols, prostaglandins, and other compounds are discussed.     

A model of the effects of heavy ion radiation on human tissue

In heavy ion radiotherapy and space travel humans are exposed to energetic heavy ions (C, Si, Fe and others). This type of irradiation often produces more severe biological effects per unit dose than more common X-rays. A new Monte Carlo model generates a physical space with the complex geometry of human tissue or a cell culture based model of tissue, which is affected by the passage of ionizing radiation. For irradiation, the model relies on a physical code for the ion track structure; for tissues, cellular maps are derived from two- or three-dimensional confocal microscopy images using image segmentation algorithm, which defines cells as pixilated volumes. The model is used to study tissue-specific statistics of direct ion hits and the remote ion action on cells. As an application of the technique, we considered the spatial pattern of apoptotic cells after heavy ion irradiation. The pattern of apoptosis is modeled as a stochastic process, which is defined by the action cross section taken from available experimental data. To characterize the degree of apoptosis, an autocorrelation function that describes the spatial correlation of apoptotic cells is introduced. The values of the autocorrelation function demonstrate the effect of the directionality of the radiation track on the spatial arrangements of inactivated cells in tissue. This effect is intrinsic only to high linear-energy-transfer radiation.     

Histologic effects of high energy electron and proton irradiation of rat brain detected with a silver-degeneration stain.

Application of the degeneration sensitive, cupric-silver staining method to brain sections of male Sprague-Dawley rats irradiated 4 days before sacrifice with 155 Mev protons, 2-8 Gy at 1 Gy/min (N=6) or 22-l0lGy at 20 Gy/min (N=16) or with 18.6 Mev electrons, 32-67 Gy at 20 Gy/min (N=20), doses which elicit behavioral changes (accelerod or conditioned taste aversion), resulted in a display of degeneration of astrocyte-like cell profiles which were not uniformly distributed. Plots of 'degeneration scores' (counts of profiles in 29 areas) vs. dose for the proton and electron irradiations displayed a linear dose response for protons in the range of 2-8 Gy. In the 20-100 Gy range, for both electrons and protons the points were distributed in a broad band suggesting a saturation curve. The dose range in which these astrocyte-like profiles becomes maximal corresponds well with the dose range for the X-ray eradication of a subtype of astrocytes, 'beta astrocytes'.     

Low fluence.

The question of the appropriate extrapolation to low dose has long been a subject of controversy. A linear no-threshold model is favored by regulatory bodies as the basis of RBE assignments and estimates of radiation hazards to the general population. This model is largely supported by extensive application of the linear-quadratic survival formula "fitted" statistically to a wide variety of experimental data obtained at doses typically exceeding 1 Gy, and then extrapolated to mGy for practical applications, and even to the prediction of hazards from single electrons. Such extrapolations are questionable at best, and may even prove hazardous for risk evaluations. Fluence and geometry rather than dose based data are proposed as a basis for a limiting "threshold" for a "low dose" extrapolation. The proposed threshold is one where the fluence of particles is one per square micron, where on average only 2/3 of the 1 micrometers2 pixels covering an irradiated area are traversed by one or more particles. The corresponding dose threshold is determined by the LET of the bombarding radiation. For relativistic electrons this dose is about 0.032 Gy.     

Mathematical model for assessment of radiation risk on long space missions.

A mathematical model is developed which describes the dynamics of radiation-induced mortality in mammalian populations. It relates statistical biometric functions with statistical characteristics and dynamics of an organism's critical system. In the framework of the model the effects of low and very low dose rates of chronic radiation on mice are simulated. Respectively, thrombocytopoietic and granulocytopoietic systems are considered as the critical ones. To calculate the dynamics of these systems, mathematical models are applied, too. In accordance with experimental data, the mortality model reproduces on quantitative level both increased and decreased mortality rates in populations of LAF1 mice, which were chronically exposed, respectively, to low and very low level radiation. All this makes it feasible to use the model as a basis for risk assessments of low level long-term irradiation.     

Can late effects of neutron irradiation in man be estimated from early responses?1

Slowly-developing tissue changes after neutron irradiation should be more easily predicted from acutely-developing injury than is the case with X rays. The difference between tissue responses to neutrons and X rays is that cell survival in both rapidly and slowly responding tissues is a direct logarithmic function of neutron dose, at least up to about 3 Gy, whereas the X-ray dose-survival relationship differs between the two types of tissue: the target cells for late injury are more susceptible to killing from accumulation of sub-lethal X-ray injury and hence the survival curve diverges from its initial essentially linear region more rapidly than does that for the target cells for acute injury.     

The effects of proton radiation on UHMWPE material properties for space flight and medical applications

Ultra High Molecular Weight Polyethylene (UHMWPE) is a polymer widely used as a radiation shielding material in space flight applications and as a bearing material in total joint replacements. As a long chain hydrocarbon based polymer, UHMWPE’s material properties are influenced by radiation exposure, and prior studies show that gamma irradiation is effective for both medical sterilization and increased wear resistance in total joint replacement applications. However, the effects of space flight radiation types and doses on UHMWPE material properties are poorly understood. In this study, three clinically relevant grades of UHMWPE (GUR 1020, GUR 1050, and GUR 1020 blended with Vitamin E) were proton irradiated and tested for differences in material properties. Each of the three types of UHMWPE was irradiated at nominal doses of 0 Gy (control), 5 Gy, 10 Gy, 20 Gy, and 35 Gy. Following irradiation, uniaxial tensile testing and thermal testing using Differential Scanning Calorimetry (DSC) and Dynamic Mechanical Analysis (DMA) were performed. Results show small but significant changes in several material properties between the control (0 Gy) and 35 Gy samples, indicating that proton irradiation could have a effect on the long term performance of UHMWPE in both medical and space flight applications.