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.     

Long-term changes in amphetamine-induced reinforcement and aversion in rats following exposure to 56Fe particle.

Exposing rats to heavy particles produces alterations in the functioning of dopaminergic neurons and in the behaviors that depend upon the integrity of the dopaminergic system. Two of these dopamine-dependent behaviors include amphetamine-induced reinforcement, measure using the conditioned place preference procedure, and amphetamine-induced reinforcement, measured using the conditioned place preference procedure, and amphetamine-induced aversion, measured using the conditioned taste aversion. Previous research has shown that exposing rats to 1.0 Gy of 1GeV/n 56Fe particles produced a disruption of an amphetamine-induced taste aversion 3 days following exposure, but produced an apparent enhancement of the aversion 112 days following exposure. The present experiments were designed to provide a further evaluation of these results by examining taste aversion learning 154 days following exposure to 1.0 Gy 56Fe particles and to establish the convergent validity of the taste aversion results by looking at the effects of exposure on the establishment of an amphetamine-induced conditioned place preference 3, 7, and 16 weeks following irradiation. The taste aversion results failed to confirm the apparent enhancement of the amphetamine-induced CTA observed in the prior experiment. However, exposure to 56Fe particles prevented the acquisition of amphetamine-induced place preference at all three-time intervals. The results are interpreted as indicating that exposure to heavy particles can produce long-term changes in behavioral functioning.     

Cognitive deficits induced by 56Fe radiation exposure.

Exposing rats to particles of high energy and charge (e.g., 56Fe) disrupts neuronal systems and the behaviors mediated by them; these adverse behavioral and neuronal effects are similar to those seen in aged animals. Because cognition declines with age, and our previous study showed that radiation disrupted Morris water maze spatial learning and memory performance, the present study used an 8-arm radial maze (RAM) to further test the cognitive behavioral consequences of radiation exposure. Control rats or rats exposed to whole-body irradiation with 1.0 Gy of 1 GeV/n high-energy 56Fe particles (delivered at the alternating gradient synchrotron at Brookhaven National Laboratory) were tested nine months following exposure. Radiation adversely affected RAM performance, and the changes seen parallel those of aging. Irradiated animals entered baited arms during the first 4 choices significantly less than did controls, produced their first error sooner, and also tended to make more errors as measured by re-entries into non-baited arms. These results show that irradiation with high-energy particles produces age-like decrements in cognitive behavior that may impair the ability of astronauts to perform critical tasks during long-term space travel beyond the magnetosphere.     

Effects of exposure to heavy particles on a behavior mediated by the dopaminergic system.

The effects of exposure to heavy particles on behaviors mediated by the central nervous system (CNS) are qualitatively different than the effects produced by exposure to other types of radiation. One behavior mediated by the CNS is the amphetamine-induced taste aversion, which is produced by pairing a novel tasting solution with injection of amphetamine. When the conditioning day is three days following irradiation, exposing rats to low doses of 56Fe particles (600 MeV/n or 1 GeV/n) eliminates the taste aversion produced by injection of amphetamine, which is dependent upon the integrity of the central dopaminergic system, but has no effect on the aversion produced by injection of lithium chloride which is mediated by the gastrointestinal system. In contrast to the effects obtained using heavy particles, exposing rats to 60CO gamma rays or to fission spectrum neutrons has no selective effect upon the acquisition of either amphetamine- or lithium chloride-induced taste aversions. When the conditioning day occurs four months following exposure to 1 GeV/n 56Fe particles, there is an enhancement of the amphetamine-induced taste aversion. The implications of these findings for approaches to risk assessment are considered.     

The effects of heavy particle irradiation on exploration and response to environmental change.

Free radicals produced by exposure to heavy particles have been found to produce motor and cognitive behavioral toxicity effects in rats similar to those found during aging. The present research was designed to investigate the effects of exposure to 56Fe particles on the ability of male Sprague-Dawley rats to detect novel arrangements in a given environment. Using a test of spatial memory previously demonstrated to be sensitive to aging, open field activity and reaction to spatial and non-spatial changes were measured in a group that received a dose of 1.5 Gy (n=10) of 56Fe heavy particle radiation or in non-radiated controls (n=10). Animals irradiated with 1.5 Gy of 56Fe particles exhibited some age-like effects in rats tested, even though they were, for the most part, subtle. Animals took longer to enter, visited less and spent significantly less time in the middle and the center portions of the open field, independently of total frequency and duration of activity of both groups. Likewise, irradiated subjects spend significantly more time exploring novel objects placed in the open field than did controls. However, irradiated subjects did not vary from controls in their exploration patterns when objects in the open field were spatially rearranged. Thus, irradiation with a dose of 1.5 Gy of 56Fe high-energy particle radiation elicited age-like effects in general open field exploratory behavior, but did not elicit age-like effects during the spatial and non-spatial rearrangement tasks.     

CNS-induced deficits of heavy particle irradiation in space: the aging connection.

Our research over the last several years has suggested that young (3 mo) rats exposed to whole-body 56Fe irradiation show neuronal signal transduction alterations and accompanying motor behavioral changes that are similar to those seen in aged (22-24 mo) rats. Since it has been postulated that 1-2% of the composition of cosmic rays contain 56Fe particles of heavy particle irradiation, there may be significant CNS effects on astronauts on long-term space flights which could produce behavioral changes that could be expressed during the mission or at some time after the return. These, when combined with other effects such as weightlessness and exposure to proton irradiations may even supercede mutagenic effects. It is suggested that by determining mechanistic relationships that might exist between aging and irradiation it may be possible to determine the common factor(s) involved in both perturbations and develop procedures to offset their deleterious effects. For example, one method that has been effective is nutritional modification.     

CNS effects of heavy particle irradiation in space: behavioral implications.

Research from several sources indicates that young (3 mo) rats exposed to heavy particle irradiation (56Fe irradiation) produces changes in motor behavior as well as alterations in neuronal transmission similar to those seen in aged (22-24 mo) rats. These changes are specific to neuronal systems that are affected by aging. Since 56Fe particles make up approximately 1-2% of cosmic rays, these findings suggest that the neuronal effects of heavy particle irradiation on long-term space flights may be significant, and may even supercede subsequent mutagenic effects in their mission capabilities. It is suggested that among other methods, it may be possible to utilize nutritional modification procedures to offset the putative deleterious effects of these particles in space.     

Operant responding following exposure to HZE particles and its relationship to particle energy and linear energy transfer

On exploratory class missions astronauts will be exposed to a variety of heavy particles (HZE particles) which differ in terms of particle energy and particle linear energy transfer. The present experiments were designed to evaluate how these physical characteristics of different particles affect cognitive performance, specifically operant responding. Following exposure to ²⁸Si, ⁴⁸Ti, ¹²C and ¹⁶O particles at the NASA Space Radiation Laboratory rats were tested for their ability to respond appropriately to changes in reinforcement schedules using an operant task. The results showed that the effectiveness of different particles in disrupting cognitive performance, defined as the lowest dose that produced a performance decrement, varied as a function of the energy of the specific particle: for comparisons between different energies of the same particle (e.g., ⁵⁶Fe) the effectiveness of the particle was directly proportional to particle linear energy transfer, whereas for comparisons between different particles (e.g., ⁵⁶Fe and ¹⁶O) effectiveness was inversely proportional to particle linear energy transfer. The results are discussed in terms of the mechanisms that influence the effectiveness of different particles and energies and in terms of their implications for analyzing the possible risks to astronauts of decrements in cognitive performance following exposure to HZE particles on long-duration exploratory class missions.     

Behavioral endpoints for radiation injury.

The relative behavioral effectiveness of heavy particles was evaluated. Using the taste aversion paradigm in rats, the behavioral toxicity of most types of radiation (including 20Ne and 40Ar) was similar to that of 60Co photons. Only 56Fe and 93Nb particles and fission neutrons were significantly more effective. Using emesis in ferrets as the behavioral endpoint, 56Fe particles and neutrons were again the most effective; however, 60Co photons were significantly more effective than 18 MeV electrons. These results suggest that LET does not completely predict behavioral effectiveness. Additionally, exposing rats to 10 cGy of 56Fe particles attenuated amphetamine-induced taste aversion learning. This behavior is one of a broad class of behaviors which depends on the integrity of the dopaminergic system and suggests the possibility of alterations in these behaviors following exposure to heavy particles in a space radiation environment.     

Effects of exposure to different types of radiation on behaviors mediated by peripheral or central systems.

The effects of exposure to ionizing radiation on behavior may result from effects on peripheral or on central systems. For behavioral endpoints that are mediated by peripheral systems (e.g., radiation-induced conditioned taste aversion or vomiting), the behavioral effects of exposure to heavy particles (56Fe, 600 MeV/n) are qualitatively similar to the effects of exposure to gamma radiation (60Co) and to fission spectrum neutrons. For these endpoints, the only differences between the different types of radiation are in terms of relative behavioral effectiveness. For behavioral endpoints that are mediated by central systems (e.g., amphetamine-induced taste aversion learning), the effects of exposure to 56Fe particles are not seen following exposure to lower LET gamma rays or fission spectrum neutrons. These results indicate that the effects of exposure to heavy particles on behavioral endpoints cannot necessarily be extrapolated from studies using gamma rays, but require the use of heavy particles.     

Early effects of low dose C ion or X-ray irradiation on human peripheral blood lymphocytes

The aim of this study was to estimate the acute effects of low dose ¹²C⁶⁺ ions or X-ray radiation on human immune function. The human peripheral blood lymphocytes (HPBL) of seven healthy donors were exposed to 0.05 Gy ¹²C⁶⁺ ions or X-ray radiation and cell responses were measured at 24 h after exposure. The cytotoxic activities of HPBL were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT); the percentages of T and NK cells subsets were detected by flow cytometry; mRNA expression of interleukin (IL)-2, tumor necrosis factor (TNF)-α and interferon (IFN)-γ were examined by real time quantitative RT-PCR (qRT-PCR); and these cytokines protein levels in supernatant of cultured cells were assayed by enzyme-linked immunosorbent assays (ELISA). The results showed that the cytotoxic activity of HPBL, mRNA expression of IL-2, IFN-γ and TNF-α in HPBL and their protein levels in supernatant were significantly increased at 24 h after exposure to 0.05 Gy ¹²C⁶⁺ ions radiation and the effects were stronger than observed for X-ray exposure. However, there was no significant change in the percentage of T and NK cells subsets of HPBL. These results suggested that 0.05 Gy high linear energy transfer (LET) ¹²C⁶⁺ radiation was a more effective approach to host immune enhancement than that of low LET X-ray. We conclude that cytokines production might be used as sensitive indicators of acute response to LDI.     

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.     

Radiosensitivity to high energy iron ions is influenced by heterozygosity for Atm, Rad9 and Brca1

Loss of function of DNA repair genes has been implicated in the development of many types of cancer. In the last several years, heterozygosity leading to haploinsufficiency for proteins involved in DNA repair was shown to play a role in genomic instability and carcinogenesis after DNA damage is induced, for example by ionizing radiation. Since the effect of heterozygosity for one gene is relatively small, we hypothesize that predisposition to cancer could be a result of the additive effect of heterozygosity for two or more genes critical to pathways that control DNA damage signaling, repair or apoptosis. We investigated the role of heterozygosity for Atm, Rad9 and Brca1 on cell oncogenic transformation and cell survival induced by 1 GeV/n⁵⁶Fe ions. Our results show that cells heterozygous for both Atm and Rad9 or Atm and Brca1 have high survival rates and are more sensitive to transformation by high energy iron ions when compared with wild-type controls or cells haploinsufficient for only one of these proteins. Since mutations or polymorphisms for similar genes exist in a small percentage of the human population, we have identified a radiosensitive sub-population. This finding has several implications. First, the existence of a radiosensitive sub-population may distort the shape of the dose–response relationship. Second, it would not be ethical to put exceptionally radiosensitive individuals into a setting where they may potentially be exposed to substantial doses of radiation.     

Antioxidant capacity reduced in scallions grown under elevated CO2 independent of assayed light intensity

Long-duration manned space missions mandate the development of a sustainable life support system and effective countermeasures against damaging space radiation. To mitigate the risk of inevitable exposure to space radiation, cultivation of fresh fruits and vegetables rich in antioxidants is an attractive alternative to pharmacological agents. However it has yet to be established whether antioxidant properties of crops can be preserved or enhanced in a space environment where environmental conditions differ from that which plants have acclimated to on earth. Scallion (Allium fistulosum) rich in antioxidant vitamins C and A, and flavonoids was used as a model plant to study the impact of a range of CO₂ concentrations and light intensities that are likely encountered in a space habitat on food quality traits. Scallions were hydroponically grown in controlled environmental chambers under a combination of 3 CO₂ concentrations of 400, 1200 and 4000 μmol mol⁻¹ and 3 light intensity levels of 150, 300, 450 μmol m⁻² s⁻¹. Total antioxidant activity (TAA) of scallion extracts was determined using a radical cation scavenging assay. Both elevated CO₂ and increasing light intensity enhanced biomass accumulation, but effects on TAA (based on dry weight) differed. TAA was reduced for plants grown under elevated CO₂, but remained unchanged with increases in light intensity. Elevated CO₂ stimulated greater biomass production than antioxidants, while an increase in photosynthetic photo flux promoted the synthesis of antioxidant compounds at a rate similar to that of biomass. Consequently light is a more effective stimulus than CO₂ for antioxidant production.     

The influence of dose, dose-rate and particle fragmentation on cataract induction by energetic iron ions.

Because activities in space necessarily involve chronic exposure to a heterogeneous charged particle radiation field it is important to assess the influence of dose-rate and the possible modulating role of heavy particle fragmentation on biological systems. Using the well-studied cataract model, mice were exposed to plateau 600 MeV/amu 56Fe ions either as acute or fractionated exposures at total doses of 5 - 504 cGy. Additional groups of mice received 20, 360 and 504 cGy behind 50 mm of polyethylene, which simulates body shielding. The reference radiation consisted of 60Co gamma radiation. The animals were examined by slit lamp biomicroscopy over their three year life spans. In accordance with our previous observations with heavy particles, the cataractogenic potential of the 600 MeV/amu 56Fe ions was greater than for low-LET radiation and increased with decreasing dose relative to gamma-rays. Fractionation of a given dose of 56Fe ions did not reduce the cataractogenicity of the radiation compared to the acute regimen. Fragmentation of the beam in the polyethylene did not alter the cataractotoxicity of the ions, either when administered singly or in fractions.     

Behavioral and neurochemical abnormalities after exposure to low doses of high-energy iron particles.

Exposure of rats to high-energy iron particles (600 MeV/amu) has been found to alter behavior after doses as low as 10 rads. The performance of a task that measures upper body strength was significantly degraded after irradiation. In addition, an impairment in the regulation of dopamine release in the caudate nucleus (a motor center in the brain), lasting at least 6 months, was also found and correlated with the performance deficits. A general indication of behavioral toxicity and an index of nausea and emesis, the conditioned taste aversion, was also evident. The sensitivity to iron particles was 10-600 times greater than to gamma photons. These results suggest that behavioral and neurobiological damage may be a consequence of exposure to low doses of heavy particles and that this possibility should be extensively studied.     

Comparison of genotoxic damage in monolayer cell cultures and three-dimensional tissue-like cell assemblies

Assessing the biological risks associated with exposure to the high-energy charged particles encountered in space is essential for the success of long-term space exploration. Although prokaryotic and eukaryotic cell models developed in our laboratory and others have advanced our understanding of many aspects of genotoxicity, in vitro models are needed to assess the risk to humans from space radiation insults. Such models must be representative of the cellular interactions present in tissues and capable of quantifying genotoxic damage. Toward this overall goal, the objectives of this study were to examine the effect of the localized microenvironment of cells, cultured as either 2-dimensional (2D) monolayers or 3-dimensional (3D) aggregates, on the rate and type of genotoxic damage resulting from exposure to Fe-charged particles, a significant portion of space radiation. We used rodent transgenic cell lines containing 50–70 copies of a LacI transgene to provide the enhanced sensitivity required to quantify mutational frequency and type in the 1100-bp LacI target as well as assessment of DNA damage to the entire 45-kbp construct. Cultured cells were exposed to high-energy Fe charged particles at Brookhaven National Laboratory’s Alternating Gradient Synchrotron facility for a total dose ranging from 0.1 to 2 Gy and allowed to recover for 0–7 days, after which mutational type and frequency were evaluated. The mutational frequency was found to be higher in 3D samples than in 2D samples at all radiation doses. Mutational frequency also was higher at 7 days after irradiation than immediately after exposure. DNA sequencing of the mutant targets revealed that deletional mutations contributed an increasingly high percentage (up to 27%) of all mutations in cells as the dose was increased from 0.5 to 2 Gy. Several mutants also showed large and complex deletions in multiple locations within the LacI target. However, no differences in mutational type were found between the 2D and the 3D samples. These 3D tissue-like model systems can reduce the uncertainty involved in extrapolating risk between in vitro cellular and in vivo models.     

Stellar β-decay rates of iron isotopes and its implications in astrophysics

β-Decay and positron decay are believed to play a consequential role during the late phases of stellar evolution of a massive star culminating in a supernova explosion. The β-decay contributes in maintaining a respectable lepton-to-baryon ratio, Y_e, of the core prior to collapse which results in a larger shock energy to produce the explosion. The positron decay acts in the opposite direction and tends to decrease the ratio. The structure of the presupernova star is altered both by the changes in Y_e and the entropy of the core material. Recently the microscopic calculation of weak interaction mediated rates on key isotopes of iron was introduced using the proton–neutron quasiparticle random phase approximation (pn-QRPA) theory with improved model parameters. Here I discuss in detail the improved calculation of β^±-decay rates for iron isotopes (^54,55,56Fe) in stellar environment. The pn-QRPA theory allows a microscopic “state-by-state” calculation of stellar rates as explained later in text. Excited state Gamow–Teller distributions are much different from ground state and a microscopic calculation of decay rates from these excited states greatly increases the reliability of the total decay rate calculation specially during the late stages of stellar evolution. The reported decay rates are also compared with earlier calculations. The positron decay rates are in reasonable agreement with the large-scale shell model calculation. The main finding of this work includes that the stellar β-decay rates of ^54,55,56Fe are around 3–5 orders of magnitude smaller than previously assumed and hence irrelevant for the determination of the evolution of Y_e during the presupernova phase of massive stars. The current work discourages the inclusion of ^55,56Fe in the list of key stellar β-decay nuclei as suggested by former simulation results.     

X-ray induced alterations in the differentiation and mineralization potential of murine preosteoblastic cells

To evaluate the effects of ionizing radiation (IR) on murine preosteoblastic cell differentiation, we directed OCT-1 cells to the osteoblastic lineage by treatment with a combination of β-glycerophosphate (β-GP), ascorbic acid (AA), and dexamethasone (Dex). In vitro mineralization was evaluated based on histochemical staining and quantification of the hydroxyapatite content of the extracellular bone matrix. Expression of mRNA encoding Runx2, transforming growth factor β1 (TGF-β1), osteocalcin (OCN), and p21^CDKN1A was analyzed. Exposure to IR reduced the growth rate and diminished cell survival of OCT-1 cells under standard conditions. Notably, calcium content analysis revealed that deposition of mineralized matrix increased significantly under osteogenic conditions after X-ray exposure in a time-dependent manner. In this study, higher radiation doses exert significant overall effects on TGF-β1, OCN, and p21^CDKN1A gene expression, suggesting that gene expression following X-ray treatment is affected in a dose-dependent manner. Additionally, we verified that Runx2 was suppressed within 24 h after irradiation at 2 and 4 Gy. Although further studies are required to verify the molecular mechanism, our observations strongly suggest that treatment with IR markedly alters the differentiation and mineralization process of preosteoblastic cells.     

Modelling of protons spectra encountered in space using medical accelerator and its microdosimetric characterization

Radiation environments in space are mainly composed of protons coming from the Galactic Cosmic Rays (GCRs) pervading the universe, the Solar Particle Events (SPEs) resulting from solar flares and coronal mass ejections, and the two Van Allen Belts surrounding the Earth due to the presence of the geomagnetic field trapping charged particles. Their wide spectra of energies up to hundreds of GeV imply diverse radiobiological effects to astronauts and radiation damage to electronics in the spacecraft. Even if lower in abundance, heavy ions such as He, C, O, Si, Fe are present in space and constitute an even bigger hazard due to their high penetrability and high linear energy transfer (LET). Most irradiation facilities available for research and testing worldwide provide usually only monoenergetic beams of high-energy protons or other heavier particles limiting studies of radiobiological effects and effects on electronics to a set of discrete energies.This paper introduces a procedure where a proton fluence spectra of interest for space radiation protection, previously generated by Monte Carlo simulations was delivered using a clinical proton therapy accelerator. Particularly, it reports the first results of modelling a proton radiation field in space in the energy range from 70 to 230 MeV during a single experimental session by programming a treatment planning system (TPS) to deliver required proton irradiation energies. Moreover, the angular distribution of the proton irradiation field has been varied to reproduce the isotropic exposure experienced by humans in space. The obtained proton radiation field was characterized using a 3D sensitive volume SOI microdosimeter developed by the Centre for Medical Radiation Physics (CMRP), University of Wollongong, Australia.