Combined Effects of Low-Dose Proton Radiation and Simulated Microgravity on the Mouse Retina and the Hematopoietic System.

The purpose of the current study was to characterize the effects of simulated microgravity and radiation-induced changes in retina and retinal vasculature, and to assess the accompanying early changes in immune cells and hematological parameters. To better understand the effects of spaceflight, we used a combination of treatments designed to simulate both the radiation and low-gravity aspects of space conditions. To simulate the broad energy spectrum of a large solar particle event (SPE) and galactic cosmic ray (GCR) radiation, male C57BL/6J mice were exposed to whole-body irradiation using fully modulated beams of 150-MeV protons containing particles of energy from 0 to 150 MeV and a uniform dose-vs.-depth profile. The mice were also hindlimb-unloaded (HLU) by tail suspension. Mice were unloaded for 7 days, exposed to 50 cGy, unloaded for an additional 7 days and then sacrificed for tissue isolation at days 4 and 30 after the combined treatments. Increases in the number of apoptotic cells were observed in the endothelial cells of mice that received radiation alone or with HLU compared to controls at both days 4 and 30 (P < 0.05). Endothelial nitric oxide synthase (eNOS) levels were significantly elevated in the retina after irradiation only or combined with HLU compared to controls at the 30-day time point (P < 0.05). The most robust changes were observed in the combination group, suggesting a synergistic response to radiation and unloading. For hematopoietic parameters, our analysis indicated the main effects for time and radiation at day 4 after treatments (day 11 postirradiation) (P < 0.05), but a smaller influence of HLU for both white blood cell and lymphocyte counts. The group treated with both radiation and HLU showed greater than 50% reduction in lymphocyte counts compared to controls. Radiation-dependent differences were also noted in specific lymphocyte subpopulations (T, B, natural killer cells). This study shows indications of an early effect of low-dose radiation and spaceflight conditions on retina and immune populations.

Acute Effect of Low-Dose Space Radiation on Mouse Retina and Retinal Endothelial Cells.

There is concern that degradation of vision as a result of space flight may compromise both mission goals and long-term quality of life after space travel. The visual disturbances may be due to a combination of intracerebral pressure changes and exposure to ionizing radiation. The retina and the retinal vasculature play important roles in vision, yet have not been studied extensively in relationship to space travel and space radiation. The goal of the current study was to characterize oxidative damage and apoptosis in retinal endothelial cells after whole-body gamma-ray, proton and oxygen (16O) ion radiation exposure at 0.1 to 1 Gy. Six-month-old male C57Bl/6J mice were whole-body irradiated with 600 MeV/n 16O ions (0, 0.1, 0.25, 1 Gy), solar particle event (SPE)-like protons (0, 0.1, 0.25, 0.5 Gy) or 60Co gamma rays (0, 0.1, 0.25, 0.5 Gy). Eyes were isolated for examining endothelial nitric oxide synthase (eNOS) expression and characterization of apoptosis in retina and retinal endothelial cells at two weeks postirradiation. The expression of eNOS was significantly increased in the retina after proton and 16O ion exposure. 16O ions induced over twofold increase in eNOS expression compared to proton exposure at two weeks postirradiation ( P < 0.05). TUNEL assays showed dose-dependent increases in apoptosis in the retina after irradiation. Low doses of 16O ions elicited apoptosis in the mouse retinal endothelial cells with the most robust changes observed after 0.1 Gy irradiation ( P < 0.05) compared to controls. Data also showed that 16O ions induced a higher frequency of apoptosis in retinal endothelial cells compared to protons ( P < 0.05). In summary, our study revealed that exposure to low-dose ionizing radiation induced oxidative damage and apoptosis in the retina. Significant changes in retinal endothelial cells occur at doses as low as 0.1 Gy. There were significant differences in the responses of endothelial cells among the radiation types examined here.

Radiation-induced alterations in synaptic neurotransmission of dentate granule cells depend on the dose and species of charged particles.

The evaluation of potential health risks associated with neuronal exposure to space radiation is critical for future long duration space travel. The purpose of this study was to evaluate and compare the effects of low-dose proton and high-energy charged particle (HZE) radiation on electrophysiological parameters of the granule cells in the dentate gyrus (DG) of the hippocampus and its associated functional consequences. We examined excitatory and inhibitory neurotransmission in DG granule cells (DGCs) in dorsal hippocampal slices from male C57BL/6 mice at 3 months after whole body irradiation with accelerated proton, silicon or iron particles. Multielectrode arrays were used to investigate evoked field synaptic potentials, an extracellular measurement of synaptic excitability in the perforant path to DG synaptic pathway. Whole-cell patch clamp recordings were used to measure miniature excitatory postsynaptic currents (mEPSCs) and miniature inhibitory postsynaptic currents (mIPSCs) in DGCs. Exposure to proton radiation increased synaptic excitability and produced dose-dependent decreases in amplitude and charge transfer of mIPSCs, without affecting the expression of γ-aminobutyric acid type A receptor α2, ß3 and γ2 subunits determined by Western blotting. Exposure to silicon radiation had no significant effects on synaptic excitability, mEPSCs or mIPSCs of DGCs. Exposure to iron radiation had no effect on synaptic excitability and mIPSCs, but significantly increased mEPSC frequency at 1 Gy, without changes in mEPSC kinetics, suggesting a presynaptic mechanism. Overall, the data suggest that proton and HZE exposure results in radiation dose- and species-dependent long-lasting alterations in synaptic neurotransmission, which could cause radiation-induced impairment of hippocampal-dependent cognitive functions.

Exposure to 56Fe-particle radiation accelerates electrophysiological alterations in the hippocampus of APP23 transgenic mice.

Abstract An unavoidable complication of space travel is exposure to high-charge, high-energy (HZE) particles. In animal studies, exposure of the CNS to HZE-particle radiation leads to neurological alterations similar to those seen in aging or Alzheimer's disease. In this study we examined whether HZE-particle radiation accelerated the age-related neuronal dysfunction that was previously described in transgenic mice overexpressing human amyloid precursor protein (APP). These APP23 transgenic mice exhibit age-related behavioral abnormalities and deficits in synaptic transmission. We exposed 7-week-old APP23 transgenic males to brain-only (56)Fe-particle radiation (600 MeV/nucleon; 1, 2, 4 Gy) and recorded synaptic transmission in hippocampal slices at 2, 6, 9, 14 and 18-24 months. We stimulated Schaeffer collaterals and recorded field excitatory postsynaptic potentials (fEPSP) and population spikes (PS) in CA1 neurons. Radiation accelerated the onset of age-related fEPSP decrements recorded at the PS threshold from 14 months of age to 9 months and reduced synaptic efficacy. At 9 months, radiation also reduced PS amplitudes. At 6 months, we observed a temporary deficit in paired-pulse inhibition of the PS at 2 Gy. Radiation did not significantly affect survival of APP23 transgenic mice. We conclude that irradiation of the brain with HZE particles accelerates Alzheimer's disease-related neurological deficits.

Dose response of CaF2:Tm to charged particles of different LET.

Thermoluminescent dosimeters are well established for performing calibrations in radiotherapy and for monitoring dose to personnel exposed to low linear energy transfer (LET) ionizing radiation. Patients undergoing light ion therapy and astronauts engaged in space flight are, however, exposed to radiation fields consisting of a mix of low- and high-LET charged particles. In this study, glow curves from CaF2:Tm chips were examined after exposure to various electron and ion beams. The annealing and readout procedures for these chips were optimized for these beams. After a 10 min prereadout annealing at 100 degrees C, the optimized glow curve samples the light output between 95 and 335 degrees C with a heating rate of 2 degrees C/s. The ratio of the integral of the glow curve under peaks 4-6 to the integral under peak 3 was approximately 0.9 for electrons, 1.0 for entrance protons, 1.6 for peak protons, and 2.2 for entrance carbon, silicon, and iron ions. The integral light output per unit dose in water for the iron exposures was about half as much as for the electron exposures. The peak-area-ratio can be used to determine a dose response factor for different LET radiations.

Bone architectural and structural properties after 56Fe26+ radiation-induced changes in body mass.

High-energy, high-charge (HZE) radiation, including iron ions ((56)Fe(26+)), is a component of the space environment. We recently observed a profound loss of trabecular bone in mice after whole-body HZE irradiation. The goal of this study was to examine morphology in bones that were excluded from a (56)Fe(26+) beam used to irradiate the body. Using 10-week-old male Sprague-Dawley rats and excluding the hind limbs and pelvis, we irradiated animals with 0, 1, 2 and 4 Gy (56)Fe(26+) ions and killed them humanely after 9 months. Animals grew throughout the experiment. Trabecular bone volume, connectivity and thickness within the proximal tibiae were significantly lower than control in a dose-dependent manner. Irradiated animals generally had less body mass than controls, which largely accounted for the variability in bone parameters as determined by ANCOVA. Likewise, lower cortical parameters were associated with reduced mass. However, lesser trabecular thickness in the 4-Gy group could not be attributed to body mass alone. Indicators of bone metabolism were generally unchanged, suggesting stabilized turnover. Exposure to (56)Fe(26+) ions can alter trabecular microarchitecture in shielded bones. Reduced body mass seems to be correlated with these deficits of trabecular and cortical bone.

A murine model for bone loss from therapeutic and space-relevant sources of radiation.

Cancer patients receiving radiation therapy are exposed to photon (gamma/X-ray), electron, and less commonly proton radiation. Similarly, astronauts on exploratory missions will be exposed to extended periods of lower-dose radiation from multiple sources and of multiple types, including heavy ions. Therapeutic doses of radiation have been shown to have deleterious consequences on bone health, occasionally causing osteoradionecrosis and spontaneous fractures. However, no animal model exists to study the cause of radiation-induced osteoporosis. Additionally, the effect of lower doses of ionizing radiation, including heavy ions, on general bone quality has not been investigated. This study presents data developing a murine model for radiation-induced bone loss. Female C57BL/6 mice were exposed to gamma, proton, carbon, or iron radiation at 2-Gray doses, representing both a clinical treatment fraction and spaceflight exposure for an exploratory mission. Mice were euthanized 110 days after irradiation. The proximal tibiae and femur diaphyses were analyzed using microcomputed tomography. Results demonstrate profound changes in trabecular architecture. Significant losses in trabecular bone volume fraction were observed for all radiation species: gamma, (-29%), proton (-35%), carbon (-39%), and iron (-34%). Trabecular connectivity density, thickness, spacing, and number were also affected. These data have clear implications for clinical radiotherapy in that bone loss in an animal model has been demonstrated at low doses. Additionally, these data suggest that space radiation has the potential to exacerbate the bone loss caused by microgravity, although lower doses and dose rates need to be studied.

Characterization of accelerated iron-ion-induced damage in gap junction-competent and -incompetent thyroid follicular cells.

Early- and late-passage cultures of Fischer rat thyroid cells differ in their growth properties and gap junction competency. Previous studies comparing early- and late-passage cultures exposed to gamma rays and proton beams revealed that differences in growth rate did not influence their responses; however, the presence of connexin 32 gap junctions conferred resistance to gamma radiation. To further assess differences in radiation quality, suspension cultures of early- and late-passage cells were exposed to accelerated iron ions, and their comparative biological responses were measured. The iron-ion-irradiated cells displayed sustained levels of incorporated dUTP, reflecting persistent DNA damage. These results were supported by the frequency of chromosomal damage measured by micronucleus formation. Iron-ion irradiation induced micronuclei at a rate of eight per gray per 100 binucleated cells scored in early-passage cells and nine per gray per 100 binucleated cells scored in late-passage cells. Relative to photons, the calculated radiobiological effectiveness for frequency of micronuclei was 5.7 and 6.4 for the early- and late-passage cultures, respectively (P > 0.05). Levels of apoptosis fluctuated as a function of dose, and modest increases above basal levels persisted throughout the 48-h period. The comparison of retained follicular structures revealed differences in the alpha components of the linear-quadratic dose-response curves (0.60 Gy(-1) for early-passage and 0.71 Gy(-1) for late-passage cultures, P < 0.014). Cell cycle phase redistribution resulted in a G2 arrest (P < 0.001) for both early- and late-passage cultures. In conclusion, the response of thyroid follicular cells to high-LET radiation was not influenced by the presence of gap junctions or the proliferative status of the target cells.

Response of thyroid follicular cells to gamma irradiation compared to proton irradiation: II. The role of connexin 32.

The objective of this study was to determine whether connexin 32-type gap junctions contribute to the "contact effect" in follicular thyrocytes and whether the response is influenced by radiation quality. Our previous studies demonstrated that early-passage follicular cultures of Fischer rat thyroid cells express functional connexin 32 gap junctions, with later-passage cultures expressing a truncated nonfunctional form of the protein. This model allowed us to assess the role of connexin 32 in radiation responsiveness without relying solely on chemical manipulation of gap junctions. The survival curves generated after gamma irradiation revealed that early-passage follicular cultures had significantly lower values of alpha (0.04 Gy(-1)) than later-passage cultures (0.11 Gy(-1)) (P < 0.0001, n = 12). As an additional way to determine whether connexin 32 was contributing to the difference in survival, cultures were treated with heptanol, resulting in higher alpha values, with early-passage cultures (0.10 Gy(-1)) nearly equivalent to untreated late-passage cultures (0.11 Gy(-1)) (P > 0.1, n = 9). This strongly suggests that the presence of functional connexin 32-type gap junctions was contributing to radiation resistance in gamma-irradiated thyroid follicles. Survival curves from proton-irradiated cultures had alpha values that were not significantly different whether cells expressed functional connexin 32 (0.10 Gy(-1)), did not express connexin 32 (0.09 Gy(-1)), or were down-regulated (early-passage plus heptanol, 0.09 Gy(-1); late-passage plus heptanol, 0.12 Gy(-1)) (P > 0.1, n = 19). Thus, for proton irradiation, the presence of connexin 32-type gap junctional channels did not influence their radiosensitivity. Collectively, the data support the following conclusions. (1) The lower alpha values from the gamma-ray survival curves of the early-passage cultures suggest greater repair efficiency and/or enhanced resistance to radiation-induced damage, coincident with the expression of connexin 32-type gap junctions. (2) The increased sensitivity of FRTL-5 cells to proton irradiation was independent of their ability to communicate through connexin 32 gap junctions. (3) The fact that the beta components of the survival curves from both gamma rays and proton beams were similar (average 0.022 +/- 0.008 Gy(-2), P > 0.1, n = 39) suggests that at higher doses the loss of viability occurs at a relatively constant rate and is independent of radiation quality and the presence of functional gap junctions.

A spontaneously arising mutation in connexin32 with repeated passage of FRTL-5 cells coincides with increased growth rate and reduced thyroxine release.

In this study we examine changes in the cellular properties of FRTL-5 cells as a function of passage number, with particular emphasis on gap junction expression, karyotype, morphology, growth rate and thyroxine (T(4)) release. Early passage FRTL-5 follicular cells transfer dye through gap junctions from injected cell(s) to third-order neighboring cells and beyond within their respective follicles and have immuno-detectable connexin32 (Cx32) type gap junctional plaques in their lateral contacting plasma membranes. By contrast, FRTL-5 cells established as monolayers, or as follicles from cultures passed more than 15 times, did not transfer microinjected Lucifer Yellow dye to contiguous neighboring cells and did not express any immuno-detectable rat thyroid specific connexins (Cx43, Cx32 or Cx26). Western blots confirmed that total, membrane and cytosolic Cx32 protein was present only in early pass follicular cultures. To better understand the passage-dependent loss of Cx32 expression, RT-PCR primers were made to the most unique sequences of the rat Cx32 molecule, the cytoplasmic and carboxyl-terminal regions. These primers were used to screen FRTL-5 RNA from cultures of various passage numbers. The results revealed that later passage cultures had a single base deletion in the middle of the Cx32 cytoplasmic loop region at nucleotide position 378. This base deletion was in the middle position of the codon for amino acid 116, which is normally a CAC (histidine) but read with the frame shift was a CCC (proline). The four amino acids that followed this deletion were also altered with the fourth one becoming UAA, the ochre translation stop codon. This premature stopping of translation resulted in a truncation of 60% of the protein, which included the remaining cytoplasmic loop, third and fourth transmembrane regions and the carboxyl-terminus. The later passage cultures did not produce a carboxyl-terminal RT-PCR product, indicating that the mRNA was also truncated. These regions of the Cx32 molecule contain the sequences and epitopes to which probes and antibodies are directed, and as such alterations of these regions with repeated passage explains reports by others that FRTL-5 cells do not express Cx32, and implies that cultures used for these assessments were passed more than 15 times. To determine if genetic or epigenetic abnormalities existed in FRTL-5 cells we performed chromosome spreads from various passage cultures. FRTL-5 cells have been reported to be diploid and more recently non-diploid; however, we found them to be fully tetraploid. This tetraploidy appears to be unstable in that later passes are tetraploid plus two or three extra chromosomes. There were no obvious translocations, breaks or large-scale interstitial deletions of any chromosomes in the FRTL-5 cultures tested. As FRTL-5 cells were repeatedly passed their morphology changed. Monolayer areas spread from beneath the follicles, and the follicles became flattened in appearance. These physical changes were coincident with dramatically increased growth rates. Early cultures (passed 3-12 times) divided on average every 49+/-1 h, whereas later passes (passes 20-25) divided every 28+/-3 h. To correlate these changes with a measure of thyroid function we assayed T(4) output. Early passage follicular cultures incubated for 6 h with sodium iodide, released on average 5.27+/- 0.33 ng/ml of T(4)/100 follicles. Later passes, or early passes treated with heptanol to down-regulate Cx32, released an average of 3.84+/-0.50 ng/ml of T(4)/100 follicles. There was a 27% difference in T(4) release between early follicular cultures, that were coupled by Cx32, and late or down-regulated early follicular cultures, that were uncoupled (P<0.0001). Collectively, the physical changes documented in this study were coincident with the loss of functional Cx32. This implies a relationship between the loss of intercellular communication and changes in morphogenic appearance, growth rate and reduced thyroid function and supports the previously postulated, tumor-suppressor role for Cx32. FRTL-5 cultures from low passage numbers are an excellent model of primary thyroid cells. However, many reports in the literature ascribe features to FRTL-5 cells that are mutually inconsistent. These differences may be resolved in the future by addressing the passage number and the conditional differences of the cultures being studied.

Dose and dose rate effects of whole-body gamma-irradiation: II. Hematological variables and cytokines.

The goal of part II of this study was to evaluate the effects of gamma-radiation on circulating blood cells, functional characteristics of splenocytes, and cytokine expression after whole-body irradiation at varying total doses and at low- and high-dose-rates (LDR, HDR). Young adult C57BL/6 mice (n = 75) were irradiated with either 1 cGy/min or 80 cGy/min photons from a 60Co source to cumulative doses of 0.5, 1.5, and 3.0 Gy. The animals were euthanized at 4 days post-exposure for in vitro assays. Significant dose- (but not dose-rate-) dependent decreases were observed in erythrocyte and blood leukocyte counts, hemoglobin, hematocrit, lipopolysaccharide (LPS)-induced 3H-thymidine incorporation, and interleukin-2 (IL-2) secretion by activated spleen cells when compared to sham-irradiated controls (p < 0.05). Basal proliferation of leukocytes in the blood and spleen increased significantly with increasing dose (p < 0.05). Significant dose rate effects were observed only in thrombocyte counts. Plasma levels of transforming growth factor-beta 1 (TGF-beta 1) and splenocyte secretion of tumor necrosis factor-alpha (TNF-alpha) were not affected by either the dose or dose rate of radiation. The data demonstrate that the responses of blood and spleen were largely dependent upon the total dose of radiation employed and that an 80-fold difference in the dose rate was not a significant factor in the great majority of measurements.

Dose and dose rate effects of whole-body gamma-irradiation: I. Lymphocytes and lymphoid organs.

The major goal of part I of this study was to compare varying doses and dose rates of whole-body gamma-radiation on lymphoid cells and organs. C57BL/6 mice (n = 75) were exposed to 0, 0.5, 1.5, and 3.0 Gy gamma-rays (60Co) at 1 cGy/min (low-dose rate, LDR) and 80 cGy/min (high-dose rate, HDR) and euthanized 4 days later. A significant dose-dependent loss of spleen mass was observed with both LDR and HDR irradiation; for the thymus this was true only with HDR. Decreasing leukocyte and lymphocyte numbers occurred with increasing dose in blood and spleen at both dose rates. The numbers (not percentages) of CD3+ T lymphocytes decreased in the blood in a dose-dependent manner at both HDR and LDR. Splenic T cell counts decreased with dose only in HDR groups; percentages increased with dose at both dose rates. Dose-dependent decreases occurred in CD4+ T helper and CD8+ T cytotoxic cell counts at HDR and LDR. In the blood the percentages of CD4+ cells increased with increasing dose at both dose rates, whereas in the spleen the counts decreased only in the HDR groups. The percentages of the CD8+ population remained stable in both blood and spleen. CD19+ B cell counts and percentages in both compartments declined markedly with increasing HDR and LDR radiation. NK1.1+ natural killer cell numbers and proportions remained relatively stable. Overall, these data indicate that the observed changes were highly dependent on the dose, but not dose rate, and that cells in the spleen are more affected by dose rate than those in blood. The results also suggest that the response of lymphocytes in different body compartments may be variable.

A comparison of mutations induced by accelerated iron particles versus those induced by low earth orbit space radiation in the FEM-3 gene of Caenorhabditis elegans.

The fem-3 gene of Caenorhabditis elegans was employed to determine the mutation frequency as well as the nature of mutations induced by low earth orbit space radiation ambient to Space Shuttle flight STS-76. Recovered mutations were compared to those induced by accelerated iron ions generated by the AGS synchrotron accelerator at Brookhaven National Laboratory. For logistical reasons, dauer larvae were prepared at TCU, transported to either Kennedy Space Center or Brookhaven National Laboratory, flown in space or irradiated, returned to TCU and screened for mutants. A total of 25 fem-3 mutants were recovered after the shuttle flight and yielded a mutation frequency of 2.1x10(-5), roughly 3.3-fold higher than the spontaneous rate of 6.3x10(-6). Four of the mutations were homozygous inviable, suggesting that they were large deletions encompassing fem-3 as well as neighboring, essential genes. Southern blot analyses revealed that one of the 25 contained a polymorphism in fem-3, further evidence that space radiation can induce deletions. While no polymorphisms were detected among the iron ion-induced mutations, three of the 15 mutants were homozygous inviable, which is in keeping with previous observations that high LET iron particles generate deficiencies. These data provide evidence, albeit indirect, that an important mutagenic component of ambient space radiation is high LET charged particles such as iron ions.

Response of thyroid follicular cells to gamma irradiation compared to proton irradiation. I. Initial characterization of DNA damage, micronucleus formation, apoptosis, cell survival, and cell cycle phase redistribution.

The RBE of protons has been assumed to be equivalent to that of photons. The objective of this study was to determine whether radiation-induced DNA and chromosome damage, apoptosis, cell killing and cell cycling in organized epithelial cells was influenced by radiation quality. Thyroid-stimulating hormone-dependent Fischer rat thyroid cells, established as follicles, were exposed to gamma rays or proton beams delivered acutely over a range of physical doses. Gamma-irradiated cells were able to repair DNA damage relatively rapidly so that by 1 h postirradiation they had approximately 20% fewer exposed 3' ends than their counterparts that had been irradiated with proton beams. The persistence of free ends of DNA in the samples irradiated with the proton beam implies that either more initial breaks or a quantitatively different type of damage had occurred. These results were further supported by an increased frequency of chromosomal damage as measured by the presence of micronuclei. Proton-beam irradiation induced micronuclei at a rate of 2.4% per gray, which at 12 Gy translated to 40% more micronuclei than in comparable gamma-irradiated cultures. The higher rate of micronucleus formation and the presence of larger micronuclei in proton-irradiated cells was further evidence that a qualitatively more severe class of damage had been induced than was induced by gamma rays. Differences in the type of damage produced were detected in the apoptosis assay, wherein a significant lag in the induction of apoptosis occurred after gamma irradiation that did not occur with protons. The more immediate expression of apoptotic cells in the cultures irradiated with the proton beam suggests that the damage inflicted was more severe. Alternatively, the cell cycle checkpoint mechanisms required for recovery from such damage might not have been invoked. Differences based on radiation quality were also evident in the alpha components of cell survival curves (0.05 Gy(-1) for gamma rays, 0.12 Gy(-1) for protons), which suggests that the higher level of survival of gamma-irradiated cells could be attributed to the persistence of nonlethally irradiated thyrocytes and/or the capacity to repair damage more effectively than cells exposed to equal physical doses of protons. The final assessment in this study was radiation-induced cell cycle phase redistribution. Gamma rays and protons produced a similar dose-dependent redistribution toward a predominantly G(2)-phase population. From our cumulative results, it seems likely that a majority of the proton-irradiated cells would not continue to divide. In conclusion, these findings suggest that there are quantitative and qualitative differences in the biological effects of proton beams and gamma rays. These differences could be due to structured energy deposition from the tracks of primary protons and the associated high-LET secondary particles produced in the targets. The results suggest that a simple dose-equivalent approach to dosimetry may be inadequate to compare the biological responses of cells to photons and protons.

Research activities at the Loma Linda University and Proton Treatment Facility--an overview.

The Loma Linda University (LLU) Radiobiology Program coordinates basic research and proton beam service activities for the university and extramural communities. The current focus of the program is on the biological and physical properties of protons and the operation of radiobiology facilities for NASA-sponsored projects. The current accelerator, supporting facilities and operations are described along with a brief review of extramural research projects supported by the program. These include space craft electronic parts and shielding testing as well as tumorigenesis and animal behavior experiments. An overview of research projects currently underway at LLU is also described. These include: 1) acute responses of the C57Bl/6 mouse immune system, 2) modulation of gene expression in the nematode C. elegans and rat thyroid cells, 3) quantitation of dose tolerance in rat CNS microvasculature, 4) behavioral screening of whole body proton and iron ion-irradiated C57Bl/6 mice, and 5) investigation of the role of cell integration into epithelial structures on responses to radiation.

Acute effects of whole-body proton irradiation on the immune system of the mouse.

The acute effects of proton whole-body irradiation on the distribution and function of leukocyte populations in the spleen and blood were examined and compared to the effects of photons derived from a (60)Co gamma-ray source. Adult female C57BL/6 mice were exposed to a single dose (3 Gy at 0.4 Gy/min) of protons at spread-out Bragg peak (SOBP), protons at the distal entry (E) region, or gamma rays and killed humanely at six different times thereafter. Specific differences were noted in the results, thereby suggesting that the kinetics of the response may be variable. However, the lack of significant differences in most assays at most times suggests that the RBE for both entry and peak regions of the Bragg curve was essentially 1.0 under the conditions of this study. The greatest immunodepression was observed at 4 days postexposure. Flow cytometry and mitogenic stimulation analyses of the spleen and peripheral blood demonstrated that lymphocyte populations differ in radiosensitivity, with B (CD19(+)) cells being most sensitive, T (CD3(+)) cells being moderately sensitive, and natural killer (NK1.1(+)) cells being most resistant. B lymphocytes showed the most rapid recovery. Comparison of the T-lymphocyte subsets showed that CD4(+) T helper/inducer cells were more radiosensitive than the CD8(+) T cytotoxic/suppressor cells. These findings should have an impact on future studies designed to maximize protection of normal tissue during and after proton-radiation exposure.

Hematological and TGF-beta variations after whole-body proton irradiation.

The acute effects of proton whole-body irradiation on five bone-marrow-derived cell types and transforming growth factor-beta 1 (TGF-beta 1) were examined and compared to the effects of photons (60Co). C57BL/6 mice were exposed to 3 Gy (0.4 Gy/min) protons at spread-out Bragg peak (SOBP), protons at entry (E), or 60Co and euthanized on days 0.5-17 thereafter. 60Co-irradiated animals had decreased erythrocytes, hemoglobin and hematocrit at 12 hours post-exposure; depression was not noted in proton (SOBP or E)-irradiated groups until day 4. Significantly decreased leukocyte counts were observed at this same time in all irradiated groups, with lymphocyte loss being greater than that of monocytes, and the depression was generally maintained. In contrast, the levels of neutrophils and thrombocytes fluctuated, especially during the first week; significant differences were noted among irradiated groups in neutrophil levels. Plasma TGF-beta 1 was elevated on day 7 in the 60Co, but not proton, irradiated mice. Collectively, the data show that dramatic and persistent changes occurred in all irradiated groups. However, few differences in assay results were seen between animals exposed to protons (SOBP or E) or photons, as well as between the groups irradiated with either of the two regions of the proton Bragg curve.

Ocular amyloidosis and secondary glaucoma.

OBJECTIVE: To report the clinical and histopathologic findings in two cases of secondary glaucoma associated with amyloidosis. DESIGN: Two case reports. METHODS: Retrospective review of clinical findings, course, and treatment of the two patients. The histopathologic findings from available biopsy material were also reviewed. MAIN OUTCOME MEASURES: Intraocular pressure (IOP), visual field changes, and surgical outcome. RESULTS: The first case describes a 76-year-old woman with orbital amyloidosis who developed gradual unilateral elevation of IOP that was poorly responsive to medical therapy and underwent filtration surgery. Episcleral venous pressure was elevated on the affected side, and histopathologic analysis of the conjunctival tissue confirmed perivascular amyloid deposits, further suggesting raised episcleral venous pressure to be a possible mechanism of glaucoma. The second case describes a 47-year-old white woman with familial amyloid neuropathy with a transthyretin cys-114 mutation. The association of glaucoma with this mutation has not been described previously. Persisting elevation of IOP in one eye was initially responsive to topical antiglaucoma medications but eventually required filtration surgery. Amyloid particles were found in the aqueous and on the lens surface. Histopathologic analysis of the aqueous and sclerectomy specimens demonstrated amyloid, suggesting outflow obstruction as a possible mechanism of glaucoma. Conjunctival buttonholing complicated filtration surgery in both cases, and the leaks eventually resolved with good control of IOP. CONCLUSIONS: Amyloid associated with glaucoma may involve different pathophysiologic mechanisms. The elevated IOP may not respond well to medical therapy. Cautious surgical manipulation of the conjunctiva is warranted in these cases.

Effects of proton and gamma radiation on lymphocyte populations and acute response to antigen.

BACKGROUND: The clinical use of proton radiation in the management of cancer, as well as benign disorders, is rapidly increasing. The major goal of this study was to compare the effects of proton and gamma (60Co) radiation on cell-mediated and humoral immunological parameters. MATERIALS AND METHODS: C57BL/6 mice were exposed to a single dose of 3 Gray (Gy) protons or gamma-rays and intraperitoneally injected 1 day later with sheep red blood cells (sRBC). On 4, 10, 15, and 29 days after exposure, subsets from each group were euthanised; nonirradiated controls (with and without sRBC injection) were included. Body and relative spleen weights, leukocyte counts, spontaneous blastogenesis, lymphocyte populations, and anti-sRBC titers were evaluated. RESULTS: The data showed significant depression (p < 0.05) in nearly all assays on days 4 and 10 after irradiation. B lymphocytes (CD19+) were the most radiosensitive, although reconstitution back to normal levels was observed by day 15. T cell (CD3+) and T helper cell (CD4+) recovery was evident by day 29, whereas the T cytotoxic cell (CD8+) count remained significantly below normal. Natural killer cells (NK1.1+) were relatively radioresistant. Anti-sRBC antibody production was slow and low titers were obtained after irradiation. No significant differences were noted between the two types of radiation. CONCLUSIONS: Taken together, the data show that whole-body irradiation with protons or gamma-rays, at the dose employed, results in marked, but transient, immunosuppression. However, at the time points of testing and with the assays used, little or no differences were found between the two forms of radiation.