Analysis of mutant quantity and quality in human-hamster hybrid AL and AL-179 cells exposed to 137Cs-gamma or HZE-Fe ions.

We measured the number of mutants and the kinds of mutations induced by 137Cs-gamma and by HZE-Fe (56Fe [600 MeV/amu, LET = 190 KeV/micrometer) in standard AL human hamster hybrid cells and in a new variant hybrid, AL-179. We found that HZE-Fe was more mutagenic than 137Cs-gamma per unit dose (about 1.6 fold), but was slightly less mutagenic per mean lethal dose, DO, at both the S1 and hprt- loci of AL cells. On the other hand, HZE-Fe induced about nine fold more complex S1- mutants than 137Cs-gamma rays, 28% vs 3%. 137Cs-gamma rays induced about twice as many S1- mutants and hprt-mutants in AL-179 as in AL cells, and about nine times more of the former were complex, and potentially unstable kinds of mutations.     

The influence of space flight factors on viability and mutability of plants.

The experiments with air-dried Crepis capillaris seeds aboard the Soyuz 16 spaceship and the orbital stations Salyut 5, 6, 7 have revealed an increase in the frequency of aberrant cells in seedlings grown from flight-exposed seeds during the flight (experiment) and after the flight on Earth (flight control) as compared to the ground-based control. The increase in seedlings grown during the flight is more significant than in the flight control. During the flight Arabidopsis thaliana developed from cotyledons to the flowering stage. Analysis of seeds setting on these plants after the flight has shown a reduction in the fertility of these plants and an increase in the frequency of recessive mutants ("Light block-1"). An increased frequency of mutants was also retained in the progeny of plants which had passed through a complete cycle of development during the flight ("Fiton-3"). Suppression of embryo viability was observed in all experiments and expressed itself in reduced germinating ability of seeds from the exposed plants and in the early death of seedlings. Damages resulting from chromosome aberrations are eliminated in the first postflight generation and damages resulting from gene mutations and micro-aberrations are preserved for a longer time.     

On the quality of mutations in mammalian cells induced by high LET radiations

The deleterious effects of accelerated heavy ions as component of the space radiation environment on living cells are of increasing importance for long duration human space flight activities. The most important aspect of such densely ionizing particle radiation is attributed to the type and quality of biological damage induced by them. This issue is addressed by investigating cell inactivation and mutation induction at the Hprt locus (coding for hypoxanthine-guanine-phosphoribosyl-transferase) of cultured V79 Chinese hamster cells exposed to densely ionizing radiation (accelerated heavy ions with different LETs from oxygen to gold, specific energies ranging from 1.9 to 69.7 MeV/u, corresponding LET values range from 62 to 13,223 keV/μm) and to sparsely ionizing radiation (200 kV X-rays). 30 spontaneous, 40 X-ray induced and 196 heavy ion induced 6-thioguanine resistant Hprt mutant colonies were characterized by Southern technique using the restriction enzymes EcoRI, PstI and BglII and a full length Hprt cDNA probe isolated from the plasmid pHPT12. Restriction patterns of the spontaneous Hprt mutants were indistinguishable from the wild type pattern, as these mutants probably contain only small deletions or even point mutations in the Hprt locus. In contrast, the overall spectrum of heavy ion induced mutations revealed a majority of partial or total deletions of the Hprt gene. With constant particle fluence (3 × 10⁶ particles/cm²) the quality of heavy ion induced mutations in the Hprt locus depends on physical parameters of the beam (atomic number, specific energy, LET). This finding suggests a relationship between the type of DNA damage and track structure. The fraction of mutants with severe deletions in the Hprt locus after exposure to oxygen ions increases from 65% at 60 keV/μm up to a maximum (100%) at 300 keV/μm and declines with higher LET values to 75% at 750 keV/μm. With heavier ions (Ca- and Au-ions) and even higher LET-values this mutant fraction decreases to 58% at 13,200 keV/μm. Heavy ion induced DNA break points in the Hprt locus are not randomly distributed.     

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.     

Preliminary results of space experiment: Implications for the effects of space radiation and microgravity on survival and mutation induction in human cells

In view of the concern for the health of astronauts that may one day journey to Mars or the Moon, we investigated the effect that space radiation and microgravity might have on DNA damage and repair. We sent frozen human lymphoblastoid TK6 cells to the International Space Station where they were maintained under frozen conditions during a 134-day mission (14 November 2008 to 28 March 2009) except for an incubation period of 8 days under 1G or μG conditions in a CO₂ incubator. The incubation period started after 100 days during which the cells had been exposed to 54 mSv of space radiation. The incubated cells were then refrozen, returned to Earth, and compared to ground control samples for the determination of the influence of microgravity on cell survival and mutation induction. The results for both varied from experiment to experiment, yielding a large SD, but the μG sample results differed significantly from the 1G sample results for each of 2 experiments, with the mean ratio of μG to 1G being 0.55 for the concentration of viable cells and 0.59 for the fraction of thymidine kinase deficient (TK⁻) mutants. Among the mutants, non-loss of zygosity events (point mutations) were less frequent (31%) after μG incubation than after 1G incubation, which might be explained by the influence of μG on cellular metabolic or physiological function. Additional experiments are needed to clarify the effect of μG interferes on DNA repair.     

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

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

Charged-particle mutagenesis II. Mutagenic effects of high energy charged particles in normal human fibroblasts.

The biological effects of high LET charged particles are a subject of great concern with regard to the prediction of radiation risk in space. In this report, mutagenic effects of high LET charged particles are quantitatively measured using primary cultures of human skin fibroblasts, and the spectrum of induced mutations are analyzed. The LET of the charged particles ranged from 25 KeV/micrometer to 975 KeV/micrometer with particle energy (on the cells) between 94-603 MeV/u. The X-chromosome linked hypoxanthine guanine phosphoribosyl transferase (hprt) locus was used as the target gene. Exposure to these high LET charged particles resulted in exponential survival curves; whereas, mutation induction was fitted by a linear model. The Relative Biological Effect (RBE) for cell-killing ranged from 3.73 to 1.25, while that for mutant induction ranged from 5.74 to 0.48. Maximum RBE values were obtained at the LET of 150 keV/micrometer. The inactivation cross-section (alpha i) and the action cross-section for mutant induction (alpha m) ranged from 2.2 to 92.0 micrometer2 and 0.09 to 5.56 x 10(-3) micrometer2, respectively. The maximum values were obtained by 56Fe with an LET of 200 keV/micrometer. The mutagenicity (alpha m/alpha i) ranged from 2.05 to 7.99 x 10(-5) with the maximum value at 150 keV/micrometer. Furthermore, molecular analysis of mutants induced by charged particles indicates that higher LET beams are more likely to cause larger deletions in the hprt locus.     

A biophysical model for estimating the frequency of radiation-induced mutations resulting from chromosomal translocations.

Gene mutations can be induced by radiation as a result of chromosomal translocations. A biophysical model is developed to estimate the frequency of this type of mutation induced by low-LET radiation. Mutations resulting from translocations are assumed to be formed by misrejoining of two DNA double strand breaks (DSB), one within the gene and one on a different chromosome. The chromosome containing the gene is assumed to occupy a spherical territory and does not overlap spatially with other chromosomes. Misrejoining between two DSB can occur only if the two DSB are closer than an interaction distance at the time of their induction. Applying the model to mutations of the hprt gene induced in G0 human lymphocyte cells by low-LET radiation, it is calculated that mutations resulting from translocations account for about 14% of the total mutations. The value of the interaction distance is determined to be 0.6 micrometers by comparing with the observed frequency of translocations in the X-chromosome.     

Molecular analysis of mutagenesis by high LET radiation.

Mutation induction by high linear energy transfer [LET] alpha particles and gamma-rays was scored in the human hamster hybrid [AL] cells. Southern blotting technique was used to analyse the molecular changes in the DNA from both the HGPRT- and S1- mutants. Dose dependent mutagenesis in the AL cells irradiated with the charged particles was higher by almost 20 fold at the S1 than the corresponding HGPRT locus. Southern analysis of the mutants induced by the high LET particles showed mostly multilocus deletion at both the HGPRT and S1 genes.     

Morphological and molecular changes of maize plants after seeds been flown on recoverablf satellite

Dry seeds of Zea mays, heterozygous for Lw₁/lw₁ alleles, sandwiched between nuclear track detectors aboard Chinese satellite for 15 days, were recovered and mutations in morphological characters on plants developed from these seeds, as well as their selected progenies, were investigated. The dosimetric results indicated that 85% of the seeds received at least 1 hit with Z≥20. About 10% of plants developed from flown seeds and 40% of observed selfed lines from the first generation plants showed some morphological changes, such as yellow stripes displayed on leaves, dwarf, anomogensis of floral organs and yellow-green seedlings, when compared with those from ground control. Using yellow stripes on leaves as the main endpoint for evaluating mutation induced in space environment, the frequency of stripe occurrence was 4.6% in the first generation plants, comparable with the results obtained from Long Duration Exposure Facility (LDEF) mission (Mei et al., 1994), but much lower than those from ground based ⁶⁰ Co-gamma treatment at a dose of 100 Gy, which reached 35.5% in the selfed lines of the second generation. One hundred and ten random primers were screened in RAPD analysis to detect the variation on genomic DNA of plants with stripes on leaves. Of these primers, 10.9% were able to generate polymorphic bands between mutated plants and control, also, common band patterns in several progenies with the same mutation phenotype were observed. These results demonstrated that space radiation environment could induce inheritable mutagenic effects on plant seeds, and verified the change in genetic material in the mutants. Further study will be needed for a better understand of the nature and mechanism of this induction of mutation.     

Mutation induction by heavy ions.

Mutation induction by heavy ions is compared in yeast and mammalian cells. Since mutants can only be recovered in survivors the influence of inactivation cross sections has to be taken into account. It is shown that both the size of the sensitive cellular site as well as track structure play an important role. Another parameter which influences the probability of mutation induction is repair: Contrary to naive assumptions primary radiation damage does not directly lead to mutations but requires modification to reconstitute the genetic machinery so that mutants can survive. The molecular structure of mutations was analyzed after exposure to deuterons by amplification with the aid of polymerase chain reaction. The results--although preliminary--demonstrate that even with densely ionizing particles a large fraction does not carry big deletions which suggests that point mutations may also be induced by heavy ions.     

Genomic instability and tumorigenic induction in immortalized human bronchial epithelial cells by heavy ions

Carcinogenesis is postulated to be a progressive multistage process characterized by an increase in genomic instability and clonal selection with each mutational event endowing a selective growth advantage. Genomic instability as manifested by the amplification of specific gene fragments is common among tumor and transformed cells. In the present study, immortalized human bronchial (BEP2D) cells were irradiated with graded doses of either 1GeV/nucleon ⁵⁶Fe ions or 150 keV/μm alpha particles. Transformed cells developed through a series of successive steps before becoming tumorigenic in nude mice. Tumorigenic cells showed neither ras mutations nor deletion in the p16 tumor suppressor gene. In contrast, they harbored mutations in the p53 gene and over-expressed cyclin D1. Genomic instability among transformed cells at various stage of the carcinogenic process was examined based on frequencies of PALA resistance. Incidence of genomic instability was highest among established tumor cell lines relative to transformed, non-tumorigenic and control cell lines. Treatment of BEP2D cells with a 4 mM dose of the aminothiol WR-1065 significantly reduced their neoplastic transforming response to ⁵⁶Fe particles. This model provides an opportunity to study the cellular and molecular mechanisms involved in malignant transformation of human epithelial cells by heavy ions.     

Spaceflight results in increase of thick filament but not thin filament proteins in the paramyosin mutant of Caenorhabditis elegans

We have investigated the effect of microgravity during spaceflight on body-wall muscle fiber size and muscle proteins in the paramyosin mutant of Caenorhabditis elegans. Both mutant and wild-type strains were subjected to 10 days of microgravity during spaceflight and compared to ground control groups. No significant change in muscle fiber size or quantity of the protein was observed in wild-type worms; where as atrophy of body-wall muscle and an increase in thick filament proteins were observed in the paramyosin mutant unc-15(e73) animals after spaceflight. We conclude that the mutant with abnormal muscle responded to microgravity by increasing the total amount of muscle protein in order to compensate for the loss of muscle function.     

LET dependence of cell death, mutation induction and chromatin damage in human cells irradiated with accelerated carbon ions.

We investigated the LET dependence of cell death, mutation induction and chromatin break induction in human embryo (HE) cells irradiated by accelerated carbon-ion beams. The results showed that cell death, mutation induction and induction of non-rejoining chromatin breaks detected by the premature chromosome condensation (PCC) technique had the same LET dependence. Carbon ions of 110 to 124keV/micrometer were the most effective at all endpoints. However, the number of initially induced chromatin breaks was independent of LET. About 10 to 15 chromatin breaks per Gy per cell were induced in the LET range of 22 to 230 keV/micrometer. The deletion pattern of exons in the HPRT locus, analyzed by the polymerase chain reaction (PCR), was LET-specific. Almost all of the mutants induced by 124 keV/micrometer beams showed deletion of the entire gene, while all mutants induced by 230keV/micrometer carbon-ion beams showed no deletion. These results suggest that the difference in the density distribution of carbon-ion track and secondary electron with various LET is responsible for the LET dependency of biological effects.     

基于NN与SVM的图像质量评价模型

为了有效地评价图像质量,利用峰值信噪比(PSNR,Pear Signal to Noise Ratio)和结构相似度(SSIM,Structure Similarity)作为图像质量的描述参数,给出"野点"的定义,提出"野点预测"并基于神经网络(NN,Neural Network)与支持向量机(SVM,Support Vector Machines)建立新的质量评价模型:神经网络用来获取质量评价映射函数,支持向量机实现样本分类.采用UTexas图像库数据进行仿真试验,质量评价模型预测图像质量的单调性比PSNR提高7.42% ,质量评价模型预测结果的均方误差平方根比PSNR提高36.06％,模型性能测试中"野点"的数目相对减少,模型性能得以提高.试验结果表明该模型的输出能有效地反映图像的主观质量.      

Mutational effects of space flight on Zea mays seeds.

The growth and development of more than 500 Zea mays seeds flown on LDEF were studied. Somatic mutations, including white-yellow stripes on leaves, dwarfing, change of leaf sheath color or seedling color were observed in plants developed from these seeds. When the frequency of white-yellow formation was used as the endpoint and compared with data from ground based studies, the dose to which maize seeds might be exposed during the flight was estimated to be equivalent to 635 cGy of gamma rays. Seeds from one particular holder gave a high mutation frequency and a wide mutation spectrum. White-yellow stripes on leaves were also found in some of the inbred progenies from plants displayed somatic mutation. Electron microscopy studies showed that the damage of chloroplast development in the white-yellow stripe on leaves was similar between seeds flown on LDEF and that irradiated by accelerated heavy ions on ground.     

The role of DNA repair on cell killing by charged particles.

It can be noted that it is not simple double strand breaks (dsb) but the non-reparable breaks that are associated with high biological effectiveness in the cell killing effect for high LET radiation. Here, we have examined the effectiveness of fast neutrons and low (initial energy = 12 MeV/u) or high (135 MeV/u) energy charged particles on cell death in 19 mammalian cell lines including radiosensitive mutants. Some of the radiosensitive lines were deficient in DNA dsb repair such as LX830, M10, V3, and L5178Y-S cells and showed lower values of relative biological effectiveness (RBE) for fast neutrons if compared with their parent cell lines. The other lines of human ataxia-telangiectasia fibroblasts, irs 1, irs 2, irs 3 and irs1SF cells, which were also radiosensitive but known as proficient in dsb repair, showed moderated RBEs. Dsb repair deficient mutants showed low RBE values for heavy ions. These experimental findings suggest that the DNA repair system does not play a major role against the attack of high linear energy transfer (LET) radiations. Therefore, we hypothesize that a main cause of cell death induced by high LET radiations is due to non-reparable dsb, which are produced at a higher rate compared to low LET radiations.     

Crop yield and light/energy efficiency in a closed ecological system: Laboratory Biosphere experiments with wheat and sweet potato.

Two crop growth experiments in the soil-based closed ecological facility, Laboratory Biosphere, were conducted from 2003 to 2004 with candidate space life support crops. Apogee wheat (Utah State University variety) was grown, planted at two densities, 400 and 800 seeds m-2. The lighting regime for the wheat crop was 16 h of light-8 h dark at a total light intensity of around 840 micromoles m-2 s-1 and 48.4 mol m-2 d-1 over 84 days. Average biomass was 1395 g m-2, 16.0 g m-2 d-1 and average seed production was 689 g m-2 and 7.9 g m-2 d-1. The less densely planted side was more productive than the denser planting, with 1634 g m-2 and 18.8 g m-2 d-1 of biomass vs. 1156 g m-2 and 13.3 g m-2 d-1; and a seed harvest of 812.3 g m-2 and 9.3 g m-2 d-1 vs. 566.5 g m-2 and 6.5 g m-2 d-1. Harvest index was 0.49 for the wheat crop. The experiment with sweet potato used TU-82-155 a compact variety developed at Tuskegee University. Light during the sweet potato experiment, on a 18 h on/6 h dark cycle, totaled 5568 total moles of light per square meter in 126 days for the sweet potatoes, or an average of 44.2 mol m-2 d-1. Temperature regime was 28 +/- 3 degrees C day/22 +/- 4 degrees C night. Sweet potato tuber yield was 39.7 kg wet weight, or an average of 7.4 kg m-2, and 7.7 kg dry weight of tubers since dry weight was about 18.6% wet weight. Average per day production was 58.7 g m-2 d-1 wet weight and 11.3 g m-2 d-1. For the wheat, average light efficiency was 0.34 g biomass per mole, and 0.17 g seed per mole. The best area of wheat had an efficiency of light utilization of 0.51 g biomass per mole and 0.22 g seed per mole. For the sweet potato crop, light efficiency per tuber wet weight was 1.33 g mol-1 and 0.34 g dry weight of tuber per mole of light. The best area of tuber production had 1.77 g mol-1 wet weight and 0.34 g mol-1 of light dry weight. The Laboratory Biosphere experiment's light efficiency was somewhat higher than the USU field results but somewhat below greenhouse trials at comparable light levels, and the best portion of the crop at 0.22 g mol-1 was in-between those values. Sweet potato production was overall close to 50% higher than trials using hydroponic methods with TU-82-155 at NASA JSC. Compared to projected yields for the Mars on Earth life support system, these wheat yields were about 15% higher, and the sweet potato yields averaged over 80% higher.     

Induction and repair of DNA strand breaks in bovine lens epithelial cells after high LET irradiation.

The lens epithelium is the initiation site for the development of radiation induced cataracts. Radiation in the cortex and nucleus interacts with proteins, while in the epithelium, experimental results reveal mutagenic and cytotoxic effects. It is suggested that incorrectly repaired DNA damage may be lethal in terms of cellular reproduction and also may initiate the development of mutations or transformations in surviving cells. The occurrence of such genetically modified cells may lead to lens opacification. For a quantitative risk estimation for astronauts and space travelers it is necessary to know the relative biological effectiveness (RBE), because the spacial and temporal distribution of initial physical damage induced by cosmic radiation differ significantly from that of X-rays. RBEs for the induction of DNA strand breaks and the efficiency of repair of these breaks were measured in cultured diploid bovine lens epithelial cells exposed to different LET irradiation to either 300 kV X-rays or to heavy ions at the UNILAC accelerator at GSI. Accelerated ions from Z=8 (O) to Z=92 (U) were used. Strand breaks were measured by hydroxyapatite chromatography of alkaline unwound DNA (overall strand breaks). Results showed that DNA damage occurs as a function of dose, of kinetic energy and of LET. For particles having the same LET the severity of the DNA damage increases with dose. For a given particle dose, as the LET rises, the numbers of DNA strand breaks increase to a maximum and then reach a plateau or decrease. Repair kinetics depend on the fluence (irradiation dose). At any LET value, repair is much slower after heavy ion exposure than after X-irradiation. For ions with an LET of less than 10,000 keV micrometers-1 more than 90 percent of the strand breaks induced are repaired within 24 hours. At higher particle fluences, especially for low energetic particles with a very high local density of energy deposition within the particle track, a higher proportion of non-rejoined breaks is found, even after prolonged periods of incubation. At the highest LET value (16,300 keV micrometers-1) no significant repair is observed. These LET-dependencies are consistent with the current mechanistic model for radiation induced cataractogenesis which postulates that genomic damage to the surviving fraction of epithelial cells is responsible for lens opacification.