Development of human epithelial cell systems for radiation risk assessment.

The most important health effect of space radiation for astronauts is cancer induction. For radiation risk assessment, an understanding of carcinogenic effect of heavy ions in human cells is most essential. In our laboratory, we have successfully developed a human mammary epithelial cell system for studying the neoplastic transformation in vitro. Growth variants were obtained from heavy ion irradiated immortal mammary cell line. These cloned growth variants can grow in regular tissue culture media and maintain anchorage dependent growth and density inhibition property. Upon further irradiation with high-LET radiation, transformed foci were found. Experimental results from these studies suggest that multiexposure of radiation is required to induce neoplastic transformation of human epithelial cells. This multihits requirement may be due to high genomic stability of human cells. These growth variants can be useful model systems for space flight experiments to determine the carcinogenic effect of space radiation in human epithelial cells.     

Oncogenic transformation of mammalian cells by ultrasoft X-rays and alpha particles.

For a better understanding of oncogenic cell transformation by ionizing radiation, we conducted experiments with ultrasoft X rays and low energy alpha particles. Confluent C3H10T1/2 cells were irradiated by Al-K (1.5 keV) X rays or alpha particles from plutonium through a thin mylar sheet, on which the cells attached and grew. Our results indicated that Al-K X rays were more effective in causing cell inactivation and oncogenic transformation than 60Co gamma rays but less effective than 1.0 and 3.7 MeV alpha particles. There was no significant difference between 1.0 and 3.7 MeV alpha particles in transforming cells although the latter were slightly more effective than the former in producing lethal effect. These results indicated that track structure is important in causing biological effects by ionizing radiation.     

Oncogenic and mutagenic effects of UV in mammalian cells

Ultraviolet light is present in the solar system and can cause major biological effects. The potential cytotoxic, mutagenic, and carcinogenic effects of UV have been studied at cellular and molecular level. Using cultured mouse embryonic fibroblasts (C3H10T1/2), we investigated the induction of mutation and transformation by UV and/or X-rays. Studies were also done with normal human mammary epithelial cells for cell inactivation and mutation induction. Curvlinear dose-response curves were observed for mutation and oncogenic transformation. The interaction between UV and X-rays depends on the sequence of exposure. When UV was given following X-irradiation, there was an additive effect. When UV was given prior to X-irradiation, however, there was a synergistic effect for both cell inactivation and transformation. The basic lesion(s) important for somatic mutation and transformation remains to be determined, and the fundamental mechanism(s) of UV and ionizing radiation interaction remains to be elucidated.     

Neoplastic cell transformation by high-LET radiation: molecular mechanisms.

Experimental data on molecular mechanisms are essential for understanding the bioeffects of radiation and for developing biophysical models, which can help in determining the shape of dose-response curves at very low doses, e.g., doses less than 1 cGy. Although it has been shown that ionizing radiation can cause neoplastic cell transformation directly, that high-LET heavy ions in general can be more effective than photons in transforming cells, and that the radiogenic cell transformation is a multi-step process [correction of processes], we know very little about the molecular nature of lesions important for cell transformation, the relationship between lethal and transformational damages, and the evolution of initial damages into final chromosomal aberrations which alter the growth control of cells. Using cultured mouse embryo cells (C3H10T1/2) as a model system, we have collected quantitative data on dose-response curves for heavy ions with various charges and energies. An analysis of these quantitative data suggested that two DNA breaks formed within 80 angstroms may cause cell transformation and that two DNA breaks formed within 20 angstroms may be lethal. Through studies with restriction enzymes which produce DNA damages at specific sites, we have found that DNA double strand breaks, including both blunt- and cohesive-ended breaks, can cause cell transformation in vitro. These results indicate that DNA double strand breaks can be important primary lesions for radiogenic cell transformation and that blunt-ended double strand breaks can form lethal as well as transformational damages due to misrepair or incomplete repair in the cell. The RBE-LET relationship is similar for HGPRT gene mutation, chromosomal deletion, and cell transformation, suggesting common lesions may be involved in these radiation effects. The high RBE of high-LET radiation for cell killing and neoplastic cell transformation is most likely related to its effectiveness in producing DNA double strand breaks in mammalian cells. At present the role of oncogenes in radiation cell transformation is unclear.     

Cellular and molecular alterations in human epithelial cells transformed by high LET radiation.

An understanding of the radiobiological effects of high LET radiation is essential for human risk estimation and radiation protection. In the present study, we show that a single, 30 cGy dose of 150 keV/micrometer 4He ions can malignantly transform human papillomavirus immortalized human bronchial epithelial [BEP2D] cells. Transformed cells produce progressively growing tumors in nude mice. The transformation frequency by the single dose of alpha particles is estimated to be approximately 4 X 10(-7). Based on the average cross-sectional area of BEP2D cells, it can be calculated that a mean traversal of 1.4 particles per cell is sufficient to induce tumorigenic conversion of these cells 3 to 4 months post-irradiation. Tumorigenic BEP2D cells overexpress mutated p53 tumor suppressor oncoproteins in addition to the cell cycle control gene cyclin D1 and D2. This model provides an opportunity to study the cellular and molecular changes at the various stages in radiation carcinogenesis involving human cells.     

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.     

Ionizing radiation-induced mutation of human cells with different DNA repair capacities.

We have observed significant differences in the response to ionizing radiation of two closely related human cells lines, and now compare the effects on these lines of both low and intermediate LET radiation. Compared to TK6, WTK1 has an enhanced X-ray survival, and is also more resistant to cell killing by alpha-particles. The hprt locus is more mutable in WTK1 than in TK6 by both X-rays and alpha-particles. WTK1 is also more mutable by alpha-particles than by X-rays at the hprt locus. X-ray-induced mutation at the heterozygous tk locus in WTK1 is about 25 fold higher than in TK6, while alpha-particle-induced mutation is nearly 50 fold higher at this locus. Also, the slowly growing tk- mutants, which comprise the majority of spontaneous and X-ray-induced tk- mutants of TK6, were not induced significantly by alpha-particles. Previously, we showed that TK6 has a reduced capacity for recombination compared with WTK1, and therefore, these results indicate that recombinational repair may contribute to both cell survival and mutation-induction following exposure to ionizing radiation. Such a mechanism may aid cell survival, but could also result in increased deleterious effects such as the unmasking of recessive mutations in cancer suppresser genes.     

The effect of space radiation of the nervous system.

The long-term effects of irradiation by accelerated heavy ions on the structure and function of the nervous system have not been studied extensively. Although the adult brain is relatively resistant to low LET radiation, cellular studies indicate that individual heavy ions can produce serious membrane lesions and multiple chromatin breaks. Capillary hemorrhages may follow high LET particle irradiation of the developing brain as high RBE effects. Evidence has been accumulating that the glial system and blood-brain barrier (BBB) are relatively sensitive to injury by ionizing radiation. While DNA repair is active in neural systems, it may be assumed that a significant portion of this molecular process is misrepair. Since the expression of cell lethality usually requires cell division, and nerve cells have an extremely low rate of division, it is possible that some of the characteristic changes of premature aging may represent a delayed effect of chromatin misrepair in brain. Altered microcirculation, decreased local metabolism, entanglement and reduction in synaptic density, premature loss of neurons, myelin degeneration, and glial proliferation are late signs of such injuries. HZE particles are very efficient in producing carcinogenic cell transformation, reaching a peak for iron particles. The promotion of viral transformation is also efficient up to an energy transfer of approximately 300 keV/micron. The RBE for carcinogenesis in nerve tissues remains unknown. On the basis of available information concerning HZE particle flux in interplanetary space, only general estimates of the magnitude of the effects of long-term spaceflight on some nervous system parameters may be constructed.     

Radiation-induced chromosomal instability in human mammary epithelial cells.

Karyotypes of human cells surviving X- and alpha-irradiation have been studied. Human mammary epithelial cells of the immortal, non-tumorigenic cell line H184B5 F5-1 M/10 were irradiated and surviving clones isolated and expanded in culture. Cytogenetic analysis was performed using dedicated software with an image analyzer. We have found that both high- and low-LET radiation induced chromosomal instability in long-term cultures, but with different characteristics. Complex chromosomal rearrangements were observed after X-rays, while chromosome loss predominated after alpha-particles. Deletions were observed in both cases. In clones derived from cells exposed to alpha-particles, some cells showed extensive chromosome breaking and double minutes. Genomic instability was correlated to delayed reproductive death and neoplastic transformation. These results indicate that chromosomal instability is a radiation-quality-dependent effect which could determine late genetic effects, and should therefore be carefully considered in the evaluation of risk for space missions.     

Recent results of comparative radiobiological experiments with short and long term expositions of arabidopsis seed embryos

Comparison of experimental data obtained from short (SDEF) and long duration exposure flights (LDEF) recently led to results, which will contribute for the estimation of genetic risk for long and/or repeated stay of man in space. Under orbital conditions biological stress and damage are induced in test subjects by cosmic radiation, especially the high energetic, densely ionizing component of heavy ions. Plant seeds were successful model systems for a biotest in studying the physiological damages and mutagenic effects caused by ionizing radiation in particular stem cells. In this article we present an overview of our space experiments with Arabidopis thaliana seeds. We present first results of investigations on certain damage endpoints (seed germination, plant survival, mutation frequencies), caused by cosmic ionizing radiation in dry dormant plant seeds of Arabidopsis thaliana after different short term (e.g. IML-1 and D-2) and long term (e.g. EURECA and LDEF-1) space exposures. Total dose effects of heavy ions and the other components of the mixed radiation field on damage endpoints and survival after space exposure and gamma-ray preirradiation were obtained. A new method of total dose spectrometry by neutron activation has been applied.     

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.     

Dose protraction studies with low- and high-LET radiations on neoplastic cell transformation in vitro.

A major objective of our heavy-ion research is to understand the potential carcinogenic effects of cosmic rays and the mechanisms of radiation-induced cell transformation. During the past several years, we have studied the relative biological effectiveness of heavy ions with various atomic numbers and linear energy transfer on neoplastic cell transformation and the repair of transformation lesions induced by heavy ions in mammalian cells. All of these studies, however, were done with a high dose rate. For risk assessment, it is extremely important to have data on the low-dose-rate effect of heavy ions. Recently, with confluent cultures of the C3H10T1/2 cell line, we have initiated some studies on the low-dose-rate effect of low- and high-LET radiation on cell transformation. For low-LET photons, there was a decrease in cell killing and cell transformation frequency when cells were irradiated with fractionated doses and at low dose rate. Cultured mammalian cells can repair both subtransformation and potential transformation lesions induced by X rays. The kinetics of potential transformation damage repair is a slow one. No sparing effect, however, was found for high-LET radiation. There was an enhancement of cell transformation for low-dose-rate argon (400 MeV/u; 120 keV/micrometer) and iron particles (600 MeV/u; 200 keV/micrometer). The molecular mechanisms for the enhancement effect is unknown at present.     

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

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

Microsatellite instability in human mammary epithelial cells transformed by heavy ions

We analyzed DNA and proteins obtained from normal and transformed human mammary epithelial cells for studying the neoplastic transformation by high-LET irradiation in vitro. We also examined microsatellite instability in human mammary cells transformed to various stages of carcinogenesis, such as normal, growth variant and tumorigenic, using microsatellite marker D5S177 on the chromosome 5 and CY17 on the Chromosome 10. Microsatellite instabilities were detected in the tumorigenic stage. These results suggest that microsatellite instability may play a role in the progression of tumorigenecity. The cause of the genomic instability has been suggested as abnormalities of DNA-repair systems which may be due to one of the three reasons: 1) alterations of cell cycle regulating genes. 2) mutations in any of the DNA mismatch repair genes. 3) mutation in any of the DNA strand breaks repair genes. No abnormality of these genes and encoded proteins, however was found in the present studies. These studies thus suggest that the microsatellite instability is induced by an alternative mechanism.     

Tumor suppressor function of Betaig-h3 gene in radiation carcinogenesis.

Interaction between cell and extracellular matrix (ECM) plays a crucial role in tumor invasiveness and metastasis. Using an immortalized human bronchial epithelial (BEP2D) cell model, we showed previously that expression of a list of genes including Betaig-h3 (induced by transforming growth factor-beta), DCC (deleted in colorectal cancer), p21(cipl), c-fos, Heat shock protein (HSP27) and cytokeratin 14 were differentially expressed in several independently generated, radiation-induced tumor cell lines (TL1-TL5) relative to parental BEP2D cells. Our previous data further demonstrated that loss of tumor suppressor gene(s) as a likely mechanism of radiation carcinogenesis. In the present study, we chose Betaig-h3 and DCC that were downregulated in tumorigenic cells for further study. Restored expression of Betaig-h3 gene, not DCC gene, by transfecting cDNA into tumor cells resulted in a significant reduction in tumor growth. While integrin receptor alpha 5 beta 1 was overexpressed in tumor cells, its expression was corrected to the level found in control BEP2D cells after Betaig-h3 transfection. These data suggest that Betaig-h3 gene is involved in tumor progression by regulating integrin alpha 5 beta 1 receptor. Furthermore, exogenous TGF- beta 1 induced expression of Betaig-h3 gene and inhibited the growth of both control and tumorigenic BEP2D cells. Therefore, downregulation of Betaig-h3 gene may results from the decreased expression of upstream mediators such as TGF-beta. The findings provide strong evidence that the Betaig-h3 gene has tumor suppressor function in radiation-induced tumorigenic human bronchial epithelial cells and suggest a potential target for interventional therapy.     

Unique radiobiological aspects of high-LET radiation.

Since the beg inning of manned space flight the potentially unique radiobiological properties of the heavy ions of the cosmic radiation had been, apart from possible interactions of radiation effects with biological effects of weightlessness, of major concern with respect to the assessment of radiation hazards in manned space flight. Radiobiological findings obtained from space flight experiments and ground based experiments with densely ionizing radiation are discussed, which suggest qualitative differences between the radiobiological mechanisms of sparsely ionizing and densely ionizing radiation. These findings comprise the observation of a long lateral range of radiobiological effectiveness around tracks of single heavy ions, the observation of micro lesions induced in biological targets by the penetration of heavy ions, the nonadditivity of radiobiological effects from sparsely and densely ionizing radiation, the different kinetics for the expression of late effects induced by sparsely or densely ionizing radiation, and the observation of a reversed dose rate effect for early and late effects induced by densely ionizing radiation. These findings bear on the radiation protection standards to be installed for a general public in manned space flight and on the design of experiments, which intend to contribute to their specification.     

Effects of heavy ions on cycling stem cells.

Murine marrow stem cells assayed with the spleen colony assay have been shown to be largely in a noncycling state, Go. In the unirradiated animal where these spleen-colony forming units (CFUs) transit normally between a non-proliferative state and active proliferation, exposure to a sufficient dose of ionizing radiation increases the frequency (probability) of this transition. For low-LET irradiation, marrow stem cells are not induced into cycle until a threshold dose is achieved. This dose appears to be in the range 50 to 100 cGy, inducing proliferation in an all-or-nothing manner. For irradiation with heavy charged-particles, however, the threshold dose is dependent on mass and energy. Irradiation with particles of sufficient mass and energy stimulates active proliferation even at the smallest doses tested, 5 cGy. Further, this response does not appear to result from an all-or-nothing effect. Rather, individual animals with intermediate levels of stem cell cycling have been observed. These data support the notion that locally controlled hemopoiesis can be affected by local deposition of radiation damage.     

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.     

Radiation biodosimetry: applications for spaceflight.

The multiparametric dosimetry system that we are developing for medical radiological defense applications could be adapted for spaceflight environments. The system complements the internationally accepted personnel dosimeters and cytogenetic analysis of chromosome aberrations, considered the best means of documenting radiation doses for health records. Our system consists of a portable hematology analyzer, molecular biodosimetry using nucleic acid and antigen-based diagnostic equipment, and a dose assessment management software application. A dry-capillary tube reagent-based centrifuge blood cell counter (QBC Autoread Plus, Becton [correction of Beckon] Dickinson Bioscience) measures peripheral blood lymphocytes and monocytes, which could determine radiation dose based on the kinetics of blood cell depletion. Molecular biomarkers for ionizing radiation exposure (gene expression changes, blood proteins) can be measured in real time using such diagnostic detection technologies as miniaturized nucleic acid sequences and antigen-based biosensors, but they require validation of dose-dependent targets and development of optimized protocols and analysis systems. The Biodosimetry Assessment Tool, a software application, calculates radiation dose based on a patient's physical signs and symptoms and blood cell count analysis. It also annotates location of personnel dosimeters, displays a summary of a patient's dosimetric information to healthcare professionals, and archives the data for further use. These radiation assessment diagnostic technologies can have dual-use applications supporting general medical-related care.     

Radiation measurements aboard NASA ER-2 high altitude aircraft with the Liulin-4J portable spectrometer.

The risks to aircrew health posed by prolonged exposure to low levels of ionizing radiation at aircraft altitudes have recently received renewed attention. Civil and military aircraft currently on the drawing board are expected to operate at higher altitudes (>12 km) and fly longer ranges than do existing aircraft, thereby exposing their crews to higher levels of ionizing radiation, for longer periods of time. We are currently carrying out dosimetric measurements of the ionizing radiation environment at approximately 20 km altitude using portable Si detectors aboard NASA's two ER-2 high altitude research aircraft. The instruments, Liulin-4J, have been extensively calibrated at several particle accelerators. With these instruments, we can measure not only absorbed dose, but also variation of the absorbed dose as a function of time. We report radiation dose measurements as function of time, altitude, and latitude for several ER-2 missions.