Radiation effects on late cytopathological parameters in the murine lens relative to particle fluence.

Lenses of mice irradiated with 250 MeV protons, 670 MeV/amu 20Ne, 600 MeV/amu 56Fe, 600 MeV/amu 93Nb and 593 MeV/amu 139La ions were evaluated by analyzing cytopathological indicators which have been implicated in the cataractogenic process. The LETs ranged from 0.40 keV/micrometer to 953 keV/micrometer and fluences from 1.31 10(3)/mm2 to 4.99 x 10(7)/mm2. 60Co gamma-rays were used as the reference radiation. The doses ranged from 10 to 40 cGy. The lenses were assessed 64 weeks post irradiation in order to observe the late effects of LET and dose on the target cell population of the lens epithelium. Our study shows that growth dependent pathological changes occur at the cellular level as a function of dose and LET. The shapes of the RBE-LET and RBE-dose curves are consistent with previous work on eye and other biological systems done in both our laboratory and others. The RBEmax's were estimated, for the most radiation cataract related cytological changes, MN frequency and MR disorganization, by calculating the ratio of the initial slopes of dose effect curve for various heavy ions to that of 60Co gamma-ray. For each ion studied, the RBEmax derived from micronucleus (MN) frequency is similar to that derived from meridional row (MR) disorganization, suggesting that heavy ions are equally efficient at producing each type of damage. Furthermore, on a per particle basis (particle/cell nucleus), both MN frequency and MR disorganization are LET dependent indicating that these classic precataractogenic indicators are multi-gene effects. Poisson probability analysis of the particle number traversing cell nuclei (average area = 24 micrometers2) suggested that single nuclear traversals determine these changes. By virtue of their precataractogenic nature the data on these endpoints intimate that radiation cataract may also be the consequence of single hits. In any case, these observations are consistent with the current theory of the mechanism of radiation cataractogenesis, which proposes that genomic damage to the epithelial cells surviving the exposure is responsible for opacification.     

Late effects from particulate radiations in primate and rabbit tissues.

Optic tissues in groups of New Zealand white rabbits were irradiated locally at different stages throughout the median life span of the species with a single dose (9 Gy) of 425 MeV/amu Ne ions (LET infinity approximately 30 keV/micrometer) and then inspected routinely for the progression of radiation cataracts. The level of early cataracts was found to be highest in the youngest group of animals irradiated (8 weeks old), but both the onset of late cataracts and loss of vision occurred earlier when animals were irradiated during the second half of the median life span. This age response can have serious implications in terms of space radiation hazards to man. Rhesus monkeys that had been subjected to whole-body skin irradiation (2.8 and 5.6 Gy) by 32 MeV protons (range in tissue approximately 1 cm) some twenty years previously were analysed for radiation damage by the propagation of skin fibroblasts in primary cultures. Such propagation from skin biopsies in MEM-alpha medium (serial cultivation) or in supplemented Ham's F-10 medium (cultivation without dilution) revealed late damage in the stem (precursor) cells of the skins of the animals. The proton fluxes employed in this experiment are representative of those occurring in major solar flares.     

Effects of gamma-ray and high energy carbon ion irradiation on swimming velocity of Euglena gracilis.

The effects of gamma-ray and high energy carbon ion irradiation on the swimming velocity of the photosynthetic flagellate Euglena gracilis strain Z were studied, focusing on a dose-effect relationship. Cells were exposed to 60Co gamma-rays at 6 doses of 10, 15, 20, 40, 100 and 200 Gy for water, and also to 290 MeV/amu carbon ions from the Heavy Ion Medical Accelerator in Chiba at 7 doses (5, 10, 15, 20, 50, 100 and 200 Gy for water). The swimming velocity was measured by a biomonitoring system, called ECOTOX. The swimming velocities of Euglena gracilis cells were significantly decreased by >40 Gy gamma-rays and >5 Gy carbon ions, respectively. The 50% effective doses for inhibition, 34 +/- 4 Gy (gamma-rays) and 13 +/- 1 Gy (290 MeV/amu carbon ions), were estimated from the best fit to data of the logistic model. The relative biological effectiveness (2.6 +/- 0.4) was calculated by the ratio of 50% effective doses. The inhibition of the swimming velocity of the cells irradiated with gamma-rays was still present after 3 days, while recovery of the swimming velocity was shown in the cells exposed to 290 MeV/amu carbon ions. It is suggested that ionizing radiation inhibits ATP production and/or increases frictional drag on beating of the flagellum, thus decreasing swimming velocity.     

Chromosome instability of HPRT-mutant subclones induced by ionising radiation of various LET.

The induction of HPRT-mutations and survival of Chinese hamster cells (line B11ii-FAF28, clone 431) were studied after irradiation by 4He and 12C-ions of various LET (20-360 keV/micrometers), produced by the U-200 heavy ion accelerator. The RBE increases with LET up to the maximum at 100-200 keV/micrometers and then decreases. Cytogenetic analysis was performed on the HPRT-mutant subclones selected from unirradiated Chinese hamster V-79 cells and from HPRT-mutant subclones that arose after exposure to gamma-rays, 1 GeV protons and 14N-ions (LET-77 keV/micrometers), produced by the synchrophasotron and the U-400M heavy ion accelerator. Slow growing mutant subclones were observed. The cytogenetic properties of individual clones were highly heterogeneous and chromosome instability was observed in both spontaneous and radiation-induced mutants. Chromosome instability was highest among spontaneous mutants and decreased with increasing LET.     

Effect of heavy ions on bacterial spores.

Inactivation of B. subtilis spores has been studied using accelerated He, C, N, O and Ne ions. The energy dependence of the inactivation cross sections for heavy ions was very weak and the mean cross sections for carbon ions (0.6-4.7 MeV/amu), nitrogen ions (0.6-4.1 MeV/amu), oxygen ions (0.8-1.1 MeV/amu), and neon ions (2.2-3.7 MeV/amu) were found to be about 0.22, 0.23, 0.26, and 0.33 micrometer2 , respectively. Analysis was carried out along lines similar to Katz's target theory but the parameters were chosen so that they have an experimental basis.     

In vivo and in vitro measurements of complex-type chromosomal exchanges induced by heavy ions.

Heavy ions are more efficient in producing complex-type chromosome exchanges than sparsely ionizing radiation, and this can potentially be used as a biomarker of radiation quality. We measured the induction of complex-type chromosomal aberrations in human peripheral blood lymphocytes exposed in vitro to accelerated H-, He-, C-, Ar-, Fe- and Au-ions in the LET range of approximately 0.4-1400 keV/micrometers. Chromosomes were analyzed either at the first post-irradiation mitosis, or in interphase, following premature condensation by phosphatase inhibitors. Selected chromosomes were then visualized after FISH-painting. The dose-response curve for the induction of complex-type exchanges by heavy ions was linear in the dose-range 0.2-1.5 Gy, while gamma-rays did not produce a significant increase in the yield of complex rearrangements in this dose range. The yield of complex aberrations after 1 Gy of heavy ions increased up to an LET around 100 keV/micrometers, and then declined at higher LET values. When mitotic cells were analyzed, the frequency of complex rearrangements after 1 Gy was about 10 times higher for Ar- or Fe- ions (the most effective ions, with LET around 100 keV/micrometers) than for 250 MeV protons, and values were about 35 times higher in prematurely condensed chromosomes. These results suggest that complex rearrangements may be detected in astronauts' blood lymphocytes after long-term space flight, because crews are exposed to HZE particles from galactic cosmic radiation. However, in a cytogenetic study of ten astronauts after long-term missions on the Mir or International Space Station, we found a very low frequency of complex rearrangements, and a significant post-flight increase was detected in only one out of the ten crewmembers. It appears that the use of complex-type exchanges as biomarker of radiation quality in vivo after low-dose chronic exposure in mixed radiation fields is hampered by statistical uncertainties.     

Chromosome aberration yields and apoptosis in human lymphocytes irradiated with Fe-ions of differing LET.

In the present paper the relationship between cell cycle delays induced by Fe-ions of differing LET and the aberration yield observable in human lymphocytes at mitosis was examined. Cells of the same donor were irradiated with 990 MeV/n Fe-ions (LET=155 keV/micrometers), 200 MeV/n Fe-ions (LET=440 keV/micrometers) and X-rays and aberrations were measured in first cycle mitoses harvested at different times after 48-84 h in culture and in prematurely condensed G2-cells (PCCs) collected at 48 h using calyculin A. Analysis of the time-course of chromosomal damage in first cycle metaphases revealed that the aberration frequency was similar after X-ray irradiation, but increased two and seven fold after exposure to 990 and 200 MeV/n Fe-ions, respectively. Consequently, RBEs derived from late sampling times were significantly higher than those obtained at early times. The PCC-data suggest that the delayed entry of heavily damaged cells into mitosis results especially from a prolonged arrest in G2. Preliminary data obtained for 4.1 MeV/n Cr-ions (LET=3160 keV/micrometers) revealed, that these delays are even more pronounced for low energy Fe-like particles. Additionally, for the different radiation qualities, BrdU-labeling indices and apoptotic indices were determined at several time-points. Only the exposure to low energy Fe-like particles affected the entry of lymphocytes into S-phase and generated a significant apoptotic response indicating that under this particular exposure condition a large proportion of heavily damaged cells is rapidly eliminated from the cell population. The significance of this observation for the estimation of the health risk associated with space radiation remains to be elucidated.     

Use of primary cell cultures to measure the late effects in the skins of rhesus monkeys irradiated with protons.

Previous pilot investigations of the uses of primary cell cultures to study late damage in stem cells of the skin of the New Zealand white (NZW) rabbit and the rhesus monkey, have been extended to individual monkeys exposed to 55 MeV protons. Protons of this energy have a larger range in tissue of (approximately 2.6 cm) than the 32 MeV protons (approximately 0.9 cm) to which the animals in our earlier studies had been exposed. Although the primary emphases in the current studies were improvement and simplification in the techniques and logistics of transportation of biopsies to a central analytical facility, comparison of the quantitative measurements obtained thus far for survival of stem cells in the skins from animals irradiated 21 years ago reveals that the effects of both proton energies are similar.     

Fluence-based relative biological effectiveness for charged particle carcinogenesis in mouse Harderian gland.

Neoplasia in the rodent Harderian gland has been used to determine the carcinogenic potential of irradiation by HZE particles. Ions from protons to lanthanum at energies up to 670 MeV/a have been used to irradiate mice, and prevalence of Harderian gland tumors has been measured 16 months after irradiation. The RBE for tumor induction has been expressed as the RBEmax, which is the ratio of the initial slopes of the dose vs prevalence curve. The RBEmax has been found to be approximately 30 for ions with LET values in excess of 100 keV/micrometer. Analysis on the basis of fluence as a substitute for dose has shown that on a per particle basis all of the ions with LET values in excess of 100 keV/micrometer have equal effectiveness. An analysis of the probabilities of ion traversals of the nucleus has shown that for these high stopping powers that a single hit is effective in producing neoplastic transformation.     

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.     

Cell cycle delays induced by heavy ion irradiation of synchronous mammalian cells.

Cell cycle effects of very high LET particles on synchronous V79 Chinese Hamster cells have been studied in a track segment experiment by means of flow cytometric methods. Cells were irradiated with 10 MeV/u Pb-ions (LET = 13500 keV/micrometers) at an average fluence of 2 particles per cell nucleus, corresponding to a survival level of about 25%. Instantaneous drastic reductions of cell proliferation in all cycle phases have been observed, which affect the cell cycle for at least 50 hours after exposure to heavy ions. These findings are in clear contrast to the results from low LET radiation experiments, where significant delays can only be observed in S-phase and G2M-phase and for comparatively short time intervals of a few hours. Additionally, high LET radiation gives rise to prolonged DNA synthesis bypassing cell division, which leads to cells with DNA content greater than that of G2M-cells.     

Damage to the photoreceptor cells of the rabbit retina from 56Fe ions: effect of age at exposure, 1.

Optic and proximate tissues of New Zealand white (NZW) rabbits at ages (approximately 3.5 years) near the middle of their median lifespan (5-7 years) were given 0.5-3.5 Gy of 465 MeV u-1 56Fe ions in the Bragg plateau region of energy deposition at a linear energy transfer (LET infinity) of 220 +/- 31 keV micrometer-1. Dose-dependent losses of retinal photoreceptor cells (rods) occurred until 1-2 years after irradiation, the period of this interim report. Similar cumulative losses of photoreceptor cells were seen during the period 1-2 years post-irradiation for rabbits given comparable exposures when young (6-9 weeks old). Since losses of photoreceptor cells at early times had not been determined previously, the current experiment, which was designed to simulate the responses of mature astronauts, redressed that deficiency.     

Inactivation, mutation induction and repair in Bacillus subtilis spores irradiated with heavy ions.

Studies on the response of bacterial spores to accelerated heavy ions (HZE particles) help in understanding problems of space radiobiology and exobiology. Layers of spores of Bacillus subtilis strains, differing in repair capabilities, were irradiated with accelerated boron, carbon and neon ions of linear energy transfer (LET) values up to 14000 MeV cm2/g. Inactivation as measured by loss of colony forming ability and induction of mutations as measured by reversion to histidine prototrophy and resistance to 150 micrograms/ml sodium azide were tested, as well as the influence of repair processes on these effects. For inactivation, the cross-sectional values sigma plotted as a function of LET follow a saturation curve. The plateau, which is reached around a LET of 2000 MeV cm2/g, occurs at 2.5 x 10(-9) cm2, a value in good agreement with the dimensions of the spore protoplast. Lethal damage produced at LET values < 2000 MeV cm2/g is reparable. Recombination repair is more effective than excision repair. At higher LET values, lethal damage could not be reconstituted by the repair mechanisms studied. In addition, at these high LET values, the frequency of induced mutations was drastically decreased. The data support the assumption of at least two qualitatively different types of lesion, depending on the LET of the affecting heavy ion.     

Neoplastic transformation of mouse C3H 10T1/2 and Syrian hamster embryo cells by heavy ions.

C3H 10T1/2 mouse-embryo fibroblasts were used for transformation experiments to study the effectiveness of various heavy ions with energies up to 20 MeV/u and LET values from 170 to 16,000 keV/micrometers. The transformation frequency per unit absorbed dose decreased with increasing ionization density; at the highest values of LET we found a decrease even of the transformation efficiency per unit fluence. Uranium ions at energies of 5, 9, and 16.3 MeV/u did not induce any transformation. In additional studies primary Syrian hamster embryo cells (SHE) were exposed to heavy ions in order to characterize cytological and molecular changes which may be correlated with neoplastic transformation. Growth behaviour, chromosomal status, tumorigenicity in nude mice, and expression of oncogenes of transformed cell lines were examined     

Cosmic ray measurements at aircraft altitudes and comparison with predictions of computer codes.

Extensive measurements of dose exposure of aircrew have been carried out in recent years using passive detectors on subsonic and supersonic air routes by DIAS (Dublin Institute for Advanced Studies). Studies were based on measurement of LET spectra using nuclear recoils produced in CR-39 nuclear track detectors by high energy neutrons and protons. The detectors were calibrated using energetic heavy ions. Data obtained were compared with the predictions of the EPCARD and CARI-6 codes. Good agreement has been found between the experimental and theoretical values.     

W-reactivation and W-mutagenesis in phage phi X 174.

W-reactivation (WR) and W-mutagenesis (WM) were observed after irradiation of phage chi X 174 with He and N ions (4.6 MeV/amu). The relative amount of phage RFI DNA (RARFI) extracted from the infected cells was determined by agarose gel electrophoresis. It was found that RARFI was increased by prior UV-irradiation of host cells and this increase corresponded to the efficiency of WR.     

DNA double strand break production and rejoining in V79 cells irradiated with light ions.

Low energy protons and other densely ionizing light ions are known to have RBE>1 for cellular end points relevant for stochastic and deterministic effects. The occurrence of a close relationship between them and induction of DNA dsb is still a matter of debate. We studied the production of DNA dsb in V79 cells irradiated with low energy protons having LET values ranging from 11 to 31 keV/micrometer, i.e. in the energy range characteristic of the Bragg peak, using the sedimentation technique. We found that the initial yield of dsb is quite insensitive to proton LET and not significantly higher than that observed with X-rays, in agreement with recent data on V79 cells irradiated with alpha particles of various LET up to 120 keV/micrometer. By contrast, RBE for cell inactivation and for mutation induction rises with the proton LET. In experiments aimed at evaluating the rejoining of dsb after proton irradiation we found that the amount of dsb left unrepaired after 120 min incubation is higher for protons than for sparsely ionizing radiation. These results indicate that dsb are not homogeneous with respect to repair and give support to the hypothesis that increasing LET leads to an increase in the complexity of DNA lesions with a consequent decrease in their repairability.     

Cataractogenesis from high-LET radiation and the Casarett model.

Space radiations, especially heavy ions, constitute significant hazards to astronauts. These hazards will increase as space missions lengthen. Moreover, the dangers to astronauts will be enhanced by the persistence, or even the progression, of biological damage throughout their subsequent life spans. To assist in the assessment of risks to astronauts, we are investigating the long-term effects of heavy ions on specific animal tissues. In one study, the eyes of rabbits of various ages were exposed to a single dose of Bragg plateau 20Ne ions (LET infinity approximately equals 30 keV/micrometer). The development of cataracts has shown a pronounced age-related response during the first year after irradiation, and will be followed for two more years. In other studies, mice were exposed to single or fractionated doses of 12C ions (4-cm spread-out Bragg peak; dose-averaged LET infinity = 70-80 keV/micrometer) or 60Co gamma-photons (LET infinity = 0.3 keV/micrometer). Measurements of the frequency of posterior lens opacification have shown that the tissue sparing observed with dose fractionation of gamma-photons was absent when 12C-ion doses were fractionated. Development of posterior lens cataracts was also followed for long periods (up to 21 months) in mice exposed to single doses of Bragg plateau HZE particles (40Ar, 20Ne and 12C ions: LET infinity approximately equals 100, 30 and 10 keV/micrometer, respectively) or 225 kVp X-rays. Based on average cataract levels at the different observation times, the RBE's (RBE = relative biological effectiveness) for the ions were circa 5, 3 and 1-2, respectively, over the range of doses used (0.05-0.9 Gy). Investigations of cataractogenesis are useful for exploring the model of radiation damage proposed by Casarett and by Rubin and Casarett with a tissue not connected directly to the vasculature.     

Induction of chromosome aberrations in mammalian cells after heavy ion exposure.

The induction of chromosome aberrations by heavy charged particles was studied in V79 Chinese hamster cells over a wide range of energies (3-100 MeV/u) and LET (20-16000 keV/micrometer). For comparison, X-ray experiments were performed. Our data indicate quantitative and qualitative differences in the response of cells to particle and x-ray irradiation. For the same level of cell survival the amount of damaged cells which can be observed is smaller in heavy ion (11.4 MeV/u Ar) irradiated samples. The highest yield of damaged cells is found 8 to 12 hours after particle irradiation and 4 hours after x-irradiation. Differences in the amount of damaged cells are attributed to cell cycle perturbations which interfere with the expression of damage. After heavy ion exposure the amount of cells reaching mitosis (mitotic index) decreases drastically and not all damaged cells reach mitosis within 48 hours after exposure. A portion of cells die in interphase. Cell cycle delays induced by x-ray irradiation are less pronounced and all cells reach the first post-irradiation mitosis within 24 hours after irradiation. Additionally, the damage produced by charged particles seems to be more severe. The disintegration of chromosomes was only observed after high LET radiation: an indication of the high and local energy deposition in the particle track. Only cross sections for the induction of chromosome aberrations in mitotic cells were reported in this paper because of the problems arising from the drastic cell cycle perturbations. In this case, cells were irradiated in mitosis and assayed immediately.     

Chromosome aberrations in human lymphocytes induced by 250 MeV protons: effects of dose, dose rate and shielding.

Although the space radiation environment consists predominantly of energetic protons, astronauts inside a spacecraft are chronically exposed to both primary particles as well as secondary particles that are generated when the primary particles penetrate the spacecraft shielding. Secondary neutrons and secondary charged particles can have an LET value that is greater than the primary protons and, therefore, produce a higher relative biological effectiveness (RBE). Using the accelerator facility at Loma Linda University, we exposed human lymphocytes in vitro to 250 MeV protons with doses ranging from 0 to 60 cGy at three different dose rates: a low dose rate of 7.5 cGy/h, an intermediate dose rate of 30 cGy/h and a high dose rate of 70 cGy/min. The effect of 15 g/cm2 aluminum shielding on the induction of chromosome aberrations was investigated for each dose rate. After exposure, lymphocytes were incubated in growth medium containing phytohemagglutinin (PHA) and chromosome spreads were collected using a chemical-induced premature chromosome condensation (PCC) technique. Aberrations were analyzed using the fluorescence in situ hybridization (FISH) technique with three different colored chromosome-painting probes. The frequency of reciprocal and complex-type chromosome exchanges were compared in shielded and unshielded samples.