Richard A. Britten

Low (20 cGy) Doses of 1 GeV/u 56Fe-Particle Radiation Lead to a Persistent Reduction in the Spatial Learning Ability of Rats

Exposure to galactic cosmic radiation (GCR) is considered to be a potential health risk in long-term space travel, and it represents a significant risk to the central nervous system (CNS). The most harmful component of GCR is the HZE [high-mass, highly charged (Z), high-energy] particles, e.g. 56Fe. In previous ground-based experiments, exposure to high doses of HZE-particle radiation induced pronounced deficits in hippocampus-dependent learning and memory in rodents. Recent data suggest that glutamatergic transmission in hippocampal synaptosomes is impaired after low (60 cGy) doses of 1 GeV/u 56Fe particles, which could lead to impairment of hippocampus-dependent spatial memory. To assess the effects of mission-relevant (20–60 cGy) doses of 1 GeV/u 56Fe particles on hippocampus-dependent spatial memory, male Wistar rats either received sham treatment or were irradiated and tested 3 months later in the Barnes maze test. Compared to the controls, rats that received 20, 40 and 60 cGy 1 GeV/u 56Fe particles showed significant impairments in their ability to locate the escape box in the Barnes maze, which was manifested by progressively increasing escape latency times over the 3 days of testing. However, this increase was not due to a lack of motivation of the rats to escape, because the total number of head pokes (and especially incorrect head pokes) remained constant over the test period. Given that rats exposed to X rays did not exhibit spatial memory impairments until >10 Gy was delivered, the RBE for 1 GeV/u 56Fe-particle-induced hippocampal spatial memory impairment is ∼50. These data demonstrate that mission-relevant doses of 1 GeV/u 56Fe particles can result in severe deficits in hippocampus-dependent neurocognitive tasks, and the extreme sensitivity of these processes to 1 GeV/u 56Fe particles must arise due to the perturbation of multiple processes in addition to killing neuronal cells.

Variations in the RBE for Cell Killing Along the Depth-Dose Profile of a Modulated Proton Therapy Beam

Considerable evidence now exists to show that that the relative biological effectiveness (RBE) changes considerably along the proton depth-dose distribution, with progressively higher RBE values at the distal part of the modulated, or spread out Bragg peak (SOBP) and in the distal dose fall-off (DDF). However, the highly variable nature of the existing studies (with regards to cell lines, and to the physical properties and dosimetry of the various proton beams) precludes any consensus regarding the RBE weighting factor at any position in the depth-dose profile. We have thus conducted a systematic study on the variation in RBE for cell killing for two clinical modulated proton beams at Indiana University and have determined the relationship between the RBE and the dose-averaged linear energy transfer (LETd) of the protons at various positions along the depth-dose profiles. Clonogenic assays were performed on human Hep2 laryngeal cancer cells and V79 cells at various positions along the SOBPs of beams with incident energies of 87 and 200 MeV. There was a marked variation in the radiosensitivity of both cell lines along the SOBP depth-dose profile of the 87 MeV proton beam. Using Hep2 cells, the D0.1 isoeffect dose RBE values (normalized against 60Co) were 1.46 at the middle of SOBP, 2.1 at the distal end of the SOBP and 2.3 in the DDF. For V79 cells, the D0.1 isoeffect RBE for the 87 MEV beam were 1.23 for the proximal end of the SOBP: 1.46 for the distal SOBP and 1.78 for the DDF. Similar D0.1 isoeffect RBE values were found for Hep2 cells irradiated at various positions along the depth-dose profile of the 200 MeV beam. Our experimentally derived RBE values were significantly correlated (P = 0.001) with the mean LETd of the protons at the various depths, which confirmed that proton RBE is highly dependent on LETd. These in vitro data suggest that the RBE of the proton beam at certain depths is greater than 1.1, a value currently used in most treatment planning algorithms. Thus, the potential for increased cell killing and normal tissue damage in the distal regions of the proton SOBP may be greater than originally thought.

Low (60 cGy) Doses of 56Fe HZE-Particle Radiation Lead to a Persistent Reduction in the Glutamatergic Readily Releasable Pool in Rat Hippocampal Synaptosomes

Abstract Exposure to galactic cosmic radiation (GCR) is considered to be a potential health risk in long-term space travel, and it represents a significant risk to the central nervous system (CNS). The most harmful component of GCR is the HZE [high-mass, highly charged (Z), high-energy] particles, e.g. 56Fe. In ground-based experiments, exposure to HZE-particle radiation induces pronounced deficits in hippocampus-dependent learning and memory in rodents. The mechanisms underlying these impairments are mostly unknown, but some studies suggest that HZE-particle exposure perturbs the regulation of long-term potentiation (LTP) at the CA1 synapse in the hippocampus. In this study, we irradiated rats with 60 cGy of 1 GeV 56Fe-particle radiation and established its impact on hippocampal glutamatergic neurotransmissions at 3 and 6 months after exposure. Exposure to 60 cGy 56Fe-particle radiation significantly (P < 0.05) reduced hyperosmotic sucrose evoked [3H]-glutamate release from hippocampal synaptosomes, a measure of the readily releasable vesicular pool (RRP). This HZE-particle-induced reduction in the glutamatergic RRP persisted for at least 6 months after exposure. At 90 days postirradiation, there was a significant reduction in the expression of the NR1, NR2A and NR2B subunits of the glutamatergic NMDA receptor. The level of the NR2A protein remained suppressed at 180 days postirradiation, but the level of NR2B and NR1 proteins returned to or exceeded normal levels, respectively. Overall, this study shows that hippocampal glutamatergic transmission is sensitive to relative low doses of 56Fe particles. Whether the observed HZE-particle-induced change in glutamate transmission, which plays a critical role in learning and memory, is the cause of HZE-particle-induced neurocognitive impairment requires further investigation.

Executive Function in Rats is Impaired by Low (20 cGy) Doses of 1 GeV/u 56Fe Particles

Exposure to galactic cosmic radiation is a potential health risk in long-term space travel and represents a significant risk to the central nervous system. The most harmful component of galactic cosmic radiation is the HZE [high mass, highly charged (Z), high energy] particles, e.g., 56Fe particle. In previous ground-based experiments, exposure to doses of HZE-particle radiation that an astronaut will receive on a deep space mission (i.e., ∼20 cGy) resulted in pronounced deficits in hippocampus-dependent learning and memory in rodents. Neurocognitive tasks that are dependent upon other regions of the brain, such as the striatum, are also impaired after exposure to low HZE-particle doses. These data raise the possibility that neurocognitive tasks regulated by the prefrontal cortex could also be impaired after exposure to mission relevant HZE-particle doses, which may prevent astronauts from performing complex executive functions. To assess the effects of mission relevant (20 cGy) doses of 1 GeV/u 56Fe particles on executive function, male Wistar rats received either sham treatment or were irradiated and tested 3 months later for their ability to perform attentional set shifting. Compared to the controls, rats that received 20 cGy of 1 GeV/u 56Fe particles showed significant impairments in their ability to complete the attentional set-shifting test, with only 17% of irradiated rats completing all stages as opposed to 78% of the control rats. The majority of failures (60%) occurred at the first reversal stage, and half of the remaining animals failed at the extra-dimensional shift phase of the studies. The irradiated rats that managed to complete the tasks did so with approximately the same ease as did the control rats. These observations suggest that exposure to mission relevant doses of 1 GeV/u 56Fe particles results in the loss of functionality in several regions of the cortex: medical prefrontal cortex, anterior cingulated cortex, posterior cingulated cortex and the basal forebrain. Our observation that 20 cGy of 1 GeV/u 56Fe particles is sufficient to impair the ability of rats to conduct attentional set-shifting raises the possibility that astronauts on prolonged deep space exploratory missions could subsequently develop deficits in executive function.

Biological Factors Influencing the RBE of Neutrons: Implications for Their Past, Present and Future Use in Radiotherapy

Abstract Britten, R. A., Peters, L. J. and Murray, D. Biological Factors Influencing the RBE of Neutrons: Implications for Their Past, Present and Future Use in Radiotherapy. Radiat. Res. 156, 125–135 (2001). The recent resurgence of interest in fast-neutron therapy, particularly for the treatment of prostate cancer, warrants a review of the original radiobiological basis for this modality and the evolution of these concepts that resulted from subsequent experimentation with the fast-neutron beams used for randomized clinical trials. It is clear from current radiobiological knowledge that some of the postulates that formed the mechanistic basis for past clinical trials were incorrect. Such discrepancies, along with the inherent physical disadvantages of neutron beams in terms of collimation and intensity modulation, may partially account for the lack of therapeutic benefit observed in many randomized clinical trials. Moreover, it is equally apparent that indiscriminate prescription of fast-neutron therapy is likely to lead to an adverse clinical outcome in a proportion of patients. Hence any renewed efforts to establish a niche for this modality in clinical radiation oncology will necessitate the development of a triage system that can discriminate those patients who might benefit from fast-neutron therapy from those who might be harmed by it. In the future, fast-neutron therapy might be prescribed based upon the relative status of appropriate molecular parameters that have a differential impact upon radiosensitivity to photons compared to fast neutrons.

Constancy of the relative biological effectiveness of 42 MeV (p→Be+) neutrons among cell lines with different DNA repair proficiencies

An important approach to understanding the role of the various DNA repair pathways in the cellular response to DNA-damaging agents is through the study of repair-deficient mutant cell lines. In the present study we used this strategy to assess the relative importance of four of these pathways for the repair of DNA damage induced by low-linear energy transfer (LET) gamma rays and intermediate-LET 42 MeV (p-->Be+) fast neutrons. The panel of hamster cell mutants that we characterized for their relative sensitivity to fast neutrons and gamma rays includes cell lines with defects in the nucleotide excision repair pathway; these can be further subdivided into mutants which are defective in nucleotide excision repair alone [UV5 (ERCC2-), UV24 (ERCC3-), UV135 (ERCC5-) and UV61 (ERCC6-)] compared to those which have an associated defect in the distinct but overlapping pathway for the repair of DNA crosslinks [UV20 (ERCC1-) and UV41 (ERCC4-)]. We also examined mutants with defects in the base excision repair pathway [EM9 (XRCC1-)] and the DNA-dependent protein kinase (DNA-PK)-mediated DNA double-strand break (DSB) repair pathway [xrs5 (XRCC5-)]. None of the mutants defective in nucleotide excision repair was differentially sensitized to fast neutrons or gamma rays; in fact, the slight radiosensitivity of these mutants under aerated conditions may be secondary to their defect in nucleotide excision repair. In contrast, deficiency in the base excision repair pathway resulted in a significant primary sensitization to both types of radiation (1.95-fold to gamma rays and 1.79-fold to neutrons). Deficiency in the DSB repair pathway mediated by DNA-PK resulted in a marked, but again similar, primary sensitization to gamma rays (4.2-fold) and neutrons (5.1-fold). Thus none of the repair pathways examined here exhibited a preferential role for the repair of damage induced by low-LET compared to intermediate-LET radiations; this resulted in an essentially constant relative biological effectiveness (RBE) of approximately 2 among the cell lines studied, independent of their DNA repair proficiency. However, consideration of these data along with data published previously for high-LET alpha particles suggests that, whereas the DNA-PK pathway is important for the repair of DSBs induced by low- and intermediate-LET radiations, it becomes less important as the LET increases beyond 100 keV/microm; thus this pathway may not be involved in repairing the more complex lesions induced by densely ionizing high-LET particles.

Exposure to Mission Relevant Doses of 1 GeV/Nucleon 56Fe Particles Leads to Impairment of Attentional Set-Shifting Performance in Socially Mature Rats

Previous ground-based experiments have shown that cranial irradiation with mission relevant (20 cGy) doses of 1 GeV/nucleon 56Fe particles leads to a significant impairment in Attentional Set Shifting (ATSET) performance, a measure of executive function, in juvenile Wistar rats. However, the use of head only radiation exposure and the biological age of the rats used in that study may not be pertinent to determine the likelihood that ATSET will be impaired in Astronauts on deep space flights. In this study we have determined the impact that whole-body exposure to 10, 15 and 20 cGy of 1 GeV/nucleon 56Fe particles had on the ability (at three months post exposure) of socially mature (retired breeder) Wistar rats to conduct the attentional set-shifting paradigm. The current study has established that whole-body exposures to 15 and 20 (but not 10) cGy of 1 GeV/nucleon 56Fe particles results in the impairment of ATSET in both juvenile and socially mature rats. However, the exact nature of the impaired ATSET performance varied depending upon the age of the rats, whether whole-body versus cranial irradiation was used and the dose of 1 GeV/u 56Fe received. Exposure of juvenile rats to 20 cGy of 1 GeV/nucleon 56Fe particles led to a decreased ability to perform intra-dimensional shifting (IDS) irrespective of whether the rats received head only or whole-body exposures. Juvenile rats that received whole-body exposure also had a reduced ability to habituate to the assay and to complete intra-dimensional shifting reversal (IDR), whereas juvenile rats that received head only exposure had a reduced ability to complete compound discrimination reversal (CDR). Socially mature rats that received whole-body exposures to 10 cGy of 1 GeV/nucleon 56Fe particles exhibited no obvious decline in set-shifting performance; however those exposed to 15 and 20 cGy had a reduced ability to perform simple discrimination (SD) and compound discrimination (CD). Exposure to 20 cGy of 1 GeV/nucleon 56Fe particles also led to a decreased performance in IDR and to ∼25% of rats failing to habituate to the task. Most of these rats started to dig for the food reward but rapidly (within 15 s) gave up digging, suggesting that they had developed appropriate procedural memories about food retrieval, but had an inability to maintain attention on the task. Our preliminary data suggests that whole-body exposure to 20 cGy of 1 GeV/nucleon 56Fe particles reduced the cholinergic (but not the GABAergic) readily releasable pool (RRP) in nerve terminals of the basal forebrain from socially-mature rats. This perturbation of the cholinergic RRP could directly lead to the loss of CDR and IDR performance, and indirectly [through the metabolic changes in the medial prefrontal cortex (mPFC)] to the loss of SD and CD performance. These findings provide the first evidence that attentional set-shifting performance in socially mature rats is impaired after whole-body exposure to mission relevant doses (15 and 20 cGy) of 1 GeV/nucleon 56Fe particles, and importantly that a dose reduction down to 10 cGy prevents that impairment. The ability to conduct Discrimination tasks (SD and CD) and reversal learning (CDR) is reduced after exposure to 15 and 20 cGy of 1 GeV/nucleon 56Fe particles, but at 20 cGy there is an additional decrement, ∼ 25% of rats are unable to maintain attention to task. These behavioral decrements are associated with a reduction in the cholinergic RRP within basal forebrain, which has been shown to play a major role in regulating the activity of the PFC.

Impaired Spatial Memory Performance in Adult Wistar Rats Exposed to Low (5–20 cGy) Doses of 1 GeV/n 56Fe Particles

Prolonged deep space missions to planets and asteroids will expose astronauts to galactic cosmic radiation, comprised of low-linear energy transfer (LET) ionizing radiations, high-energy protons and high-Z and energy (HZE) particles, such as 56Fe nuclei. In prior studies with rodents exposed to HZE particle radiation at doses likely to be encountered during deep space missions (<20 cGy) investigators reported impaired hippocampal-dependent neurocognitive performance and further observed substantial variation among the irradiated animals in neurocognitive impairment, ranging from no observable effects to severe impairment. These findings point to the importance of incorporating quantitative measures of interindividual variations into next generation risk assessment models of radiation risks on neurocognition. In this study, 269 male proven breeder Wistar rats were exposed to 1 GeV/n 56Fe at doses of 0, 5, 10, 15 and 20 cGy, and tested for spatial memory performance on the Barnes maze at three months after exposure. The radiation response data were compared using changes in mean cohort performance and by the proportion of poor responders using the performance benchmark of two standard deviations below the mean value among the sham-irradiated cohort. Acute exposures to mission-relevant doses of 1 GeV/n 56Fe reduced the mean spatial memory performance at three months after exposure (P < 0.002) and increased the proportions of poor performers, 2- to 3-fold. However, a substantial fraction of animals in all exposure cohorts showed no detectable change in performance, compared to the distribution of sham-irradiated animals. Our findings suggest that individualized metrics of susceptibility or resistance to radiation-induce changes in neurocognitive performance will be advantageous to the development of probabilistic risk assessment models for HZE-induced neurocognitive impairment.

The Influence of Hypoxia on the Relative Sensitivity of Human Tumor Cells to 62.5 MeV (p→Be) Fast Neutrons and 4 MeV Photons

Abstract Warenius, H. M., White, R., Peacock, J. H., Hanson, J., Britten, R. A. and Murray, D. The Influence of Hypoxia on the Relative Sensitivity of Human Tumor Cells to 62.5 MeV (p→Be) Fast Neutrons and 4 MeV Photons. Fast neutrons have been used in the clinical radiation therapy of tumors largely because of experimental evidence that their cytotoxic effects are much less dependent on oxygen levels than those of low-LET photons. The potential therapeutic advantage of fast neutrons based on hypoxia alone can be calculated as the “hypoxic gain factor”, which is the ratio of the OERs for the fast-neutron compared to the photon beams. The hypoxic gain factor that is generally anticipated based on studies with established mammalian cell lines is about 1.6. However, surprisingly few studies have examined the influence of hypoxia on the fast-neutron radiosensitivity of human tumor cells of different histological types. For this reason, we have determined the OERs of five human tumor cell lines exposed to 62.5 MeV (p→Be) cyclotron-generated fast neutrons or 4 MeV photons from a clinical linear accelerator. The OERs for four chemotherapy-naive cell lines, HT29/5, Hep2, HeLa and RT112, were invariably greater for photons than for neutrons, but all of these values were lower than expected on the basis of the previous literature. Despite their low OERs, these cell lines showed hypoxic gain factors that were within the range of 1.31−1.63, indicating that such effects cannot entirely explain the disappointing clinical results obtained with fast neutrons. In contrast, comparison of the surviving fractions at clinically relevant doses (1.6 Gy of neutrons and 2.0 Gy of photons) for these four tumor cell lines suggested that little benefit should result from neutron treatment. Only the cisplatin-resistant OAW42-CP line showed a significant hypoxic gain factor by this method of analysis. We conclude that, at the dose fractions used in clinical radiation therapy, there may not be a radiobiological precedent for higher local control rates after fast-neutron irradiation of hypoxic tumor cells.

Exposure to Mission-Relevant Doses of 1 GeV/n 48Ti Particles Impairs Attentional Set-Shifting Performance in Retired Breeder Rats

Astronauts on deep space missions will be required to work more autonomously than on previous missions, and thus their ability to perform executive functions could be critical to mission success. In this study we have determined the impact that exposure to 10, 15 and 20 cGy of 1 GeV/n 48Ti particles has on the long-term (three-months post exposure) ability of male retired breeder Wistar rats to perform attentional set shifting. The ability of the rats to conduct compound discrimination reversal (CDR) was significantly impaired at all doses studied, with compound discrimination (CD) being impaired at 10 and 15 cGy. Impaired CD performance would result in a decreased ability to identify and focus on relevant aspects of a task being conducted, while the functional consequence of an impaired CDR performance would be a reduction in the individuals ability to recognize when that factor changes from a positive to a negative factor for the successful completion of a task. In contrast to our previous study with 1 GeV/n 56Fe particles, there were no significant impairments in the ability of the 48Ti-irradiated rats to conduct simple discrimination. This study further supports the notion that “mission-relevant” doses of HZE particles (<20 cGy) can impair certain aspects of attentional set-shifting performance in retired breeder rats, but there may be some ion-specific changes in the specific cognitive domains impaired.