Enzymic chemical reaction under microgravity environment in space

In recent years, some papers have reported synergism in the biological effects of space radiation and microgravity. However, there is no direct evidence for these phenomena. As one possible mechanism, we investigated whether DNA ligation in the final step of DSBs repair of DNA molecules induced by radiation is depressed by microgravity. Therefore, we have scheduled the space experiments of the effects of microgravity on repair activity of T4 DNA ligase for DSBs prepared with digestion of a restriction enzyme (Sma I) to plasmid DNA. As another possible mechanism, the high mutation frequency may be induced from abnormal base-incorporation during DNA replication under microgravity. Using the Taq polymerase and polymerase III, we have also scheduled whether mutation frequency is affected by microgravity during DNA replication for a damaged DNA base induced by an alkylating agent (N-methyl-N-nitrosourea, MNU).     

Impact of microgravity on radiobiological processes and efficiency of DNA repair

To study the influence of microgravity on radiobiological processes in space, space experiments have been performed, using an on-board 1×g reference centrifuge as in-flight control. The trajectory of individual heavy ions was localized in relation to the biological systems by use of the Biostack concept, or an additional high dose of radiation was applied either before the mission or during the mission from an on-board radiation source. In embryonic systems, such as early developmental stages of Drosophila melanogaster and Carausius morosus, the occurrence of chromosomal translocations and larval malformations was dramatically increased in response to microgravity and radiation. It has been hypothesized that these synergistic effects might be caused by an interference of microgravity with DNA repair processes. However, recent studies on bacteria, yeast cells and human fibroblasts suggest that a disturbance of cellular repair processes in the microgravity environment might not be a complete explanation for the reported synergism of radiation and microgravity. As an alternative explanation, an impact of microgravity on signal transduction, on the metabolic/physiological state or on the chromatin structure at the cellular level, or modification of self-assembly, intercellular communication, cell migration, pattern formation or differentiation at the tissue and organ level should be considered.     

In vitro cultured cells as probes for space radiation effects on biological systems

Near future scenarios of long-term and far-reaching manned space missions, require more extensive knowledge of all possible biological consequences of space radiation, particularly in humans, on both a long-term and a short-term basis. In vitro cultured cells have significantly contributed to the tremendous advancement of biomedical research. It is therefore to be expected that simple biological systems such as cultured cells, will contribute to space biomedical sciences. Space represents a novel environment, to which life has not been previously exposed. Both microgravity and space radiation are the two relevant components of such an environment, but biological adaptive mechanisms and efficient countermeasures can significantly minimize microgravity effects. On the other hand, it is felt that space radiation risks may be more relevant and that defensive strategies can only stem from our deeper knowledge of biological effects and of cellular repair mechanisms. Cultured cells may play a key role in such studies. Particularly, thyroid cells may be relevant because of the exquisite sensitivity of the thyroid gland to radiation. In addition, a clone of differentiated, normal thyroid follicular cells (FRTL5 cells) is available in culture, which is well characterized and particularly fit for space research.     

The biological effects of space radiation during long stays in space.

Many space experiments are scheduled for the International Space Station (ISS). Completion of the ISS will soon become a reality. Astronauts will be exposed to low-level background components from space radiation including heavy ions and other high-linear energy transfer (LET) radiation. For long-term stay in space, we have to protect human health from space radiation. At the same time, we should recognize the maximum permissible doses of space radiation. In recent years, physical monitoring of space radiation has detected about 1 mSv per day. This value is almost 150 times higher than that on the surface of the Earth. However, the direct effects of space radiation on human health are currently unknown. Therefore, it is important to measure biological dosimetry to calculate relative biological effectiveness (RBE) for human health during long-term flight. The RBE is possibly modified by microgravity. In order to understand the exact RBE and any interaction with microgravity, the ISS centrifugation system will be a critical tool, and it is hoped that this system will be in operation as soon as possible.     

Proposal from space radiation biologists. Importance of Centrifuge Facility in the study of biological effect by space radiation]

In microgravity, astronauts were constantly exposed to space radiation containing various kinds of radiation with a low-dose rate during long-term stays in space. It is very difficult to define the relative biological effectiveness (RBE) of space radiation under microgravity. In order to understand correct the RBE of space radiation, therefore, utilization of Centrifuge Facility is desired as a control experiment at orbit for removing other factors such as microgravity except space radiation. Here, we summarized the importance of Centrifuge Facility in the study of biological effect of space radiation.     

Radiation response mechanisms of the extremely radioresistant bacterium Deinococcus radiodurans.

Effect of microgravity on recovery of bacterial cells from radiation damage was examined in IML-2, S/MM-4 and S/MM-9 experiments using the extremely radioresistant bacterium Deinococcus radiodurans. The cells were irradiated with gamma rays before the space flight and incubated on board the Space Shuttle. The survival of the wild type cells incubated in space increased compared with the ground controls, suggesting that the recovery of this bacterium from radiation damage was enhanced under the space environment. No difference was observed between the survivals of radiosensitive mutant rec30 cells incubated in space and on the ground. The amount of DNA-repair related RecA protein induced under microgravity was similar to those of ground controls, however, induction of PprA protein, product of a unique radiation-inducible gene (designated pprA) responsible for loss of radiation resistance in repair-deficient mutant, KH311, was enhanced under microgravity compared with ground controls. Recent investigation in vitro showed that PprA preferentially bound to double-stranded DNA carrying strand breaks, inhibited Escherichia coli exonuclease III activity, and stimulated the DNA end-joining reaction catalyzed by DNA ligases. These results suggest that D. radiodurans has a radiation-induced non-homologous end-joining (NHEJ) repair mechanism in which PprA plays a critical role.     

Simulated microgravity decreases DNA repair capacity and induces DNA damage in human lymphocytes

The effect of simulated microgravity on DNA damage and apoptosis is still controversial. The objective of this study was to test whether simulated microgravity conditions affect the expression of genes for DNA repair and apoptosis. To achieve this objective, human lymphocyte cells were grown in a NASA‐developed rotating wall vessel (RWV) bioreactor that simulates microgravity. The same cell line was grown in parallel under normal gravitational conditions in culture flasks. The effect of microgravity on the expression of genes was measured by quantitative real‐time PCR while DNA damage was examined by comet assay. The result of this study revealed that exposure to simulated microgravity condition decreases the expression of DNA repair genes. Mismatch repair (MMR) class of DNA repair pathway were more susceptible to microgravity condition‐induced gene expression changes than base excision repair (BER) and nucleotide excision repair (NER) class of DNA repair genes. Downregulation of genes involved in cell proliferation (CyclinD1 and PCNA) and apoptosis (Bax) was also observed. Microgravity‐induced changes in the expression of some of these genes were further verified at the protein level by Western blot analysis. The findings of this study suggest that microgravity may induce alterations in the expression of these DNA repair genes resulting in accumulation of DNA damage. Reduced expression of cell‐cycle genes suggests that microgravity may cause a reduction in cell growth. Downregulation of pro‐apoptotic genes further suggests that extended exposure to microgravity may result in a reduction in the cells' ability to undergo apoptosis. Any resistance to apoptosis seen in cells with damaged DNA may eventually lead to malignant transformation of those cells. J. Cell. Biochem. 107: 723–731, 2009. © 2009 Wiley‐Liss, Inc.     

Characterization of Epstein–Barr virus reactivation in a modeled spaceflight system

Epstein–Barr virus (EBV) is the causative agent of mononucleosis and is also associated with several malignancies, including Burkitt's lymphoma, Hodgkin's lymphoma, and nasopharyngeal carcinoma, among others. EBV reactivates during spaceflight, with EBV shedding in saliva increasing to levels ten times those observed pre‐and post‐flight. Although stress has been shown to increase reactivation of EBV, other factors such as radiation and microgravity have been hypothesized to contribute to reactivation in space. We used a modeled spaceflight environment to evaluate the influence of radiation and microgravity on EBV reactivation. BJAB (EBV‐negative) and Raji (EBV‐positive) cell lines were assessed for viability/apoptosis, viral antigen and reactive oxygen species expression, and DNA damage and repair. EBV‐infected cells did not experience decreased viability and increased apoptosis due to modeled spaceflight, whereas an EBV‐negative cell line did, suggesting that EBV infection provided protection against apoptosis and cell death. Radiation was the major contributor to EBV ZEBRA upregulation. Combining modeled microgravity and radiation increased DNA damage and reactive oxygen species while modeled microgravity alone decreased DNA repair in Raji cells. Additionally, EBV‐infected cells had increased DNA damage compared to EBV‐negative cells. Since EBV‐infected cells do not undergo apoptosis as readily as uninfected cells, it is possible that virus‐infected cells in EBV seropositive individuals may have an increased risk to accumulate DNA damage during spaceflight. More studies are warranted to investigate this possibility. J. Cell. Biochem. 114: 616–624, 2013. © 2012 Wiley Periodicals, Inc.     

"Modeled microgravity" affects cell response to ionizing radiation and increases genomic damage

The aim of this work was to assess whether "modeled microgravity" affects cell response to ionizing radiation, increasing the risk associated with radiation exposure. Lymphoblastoid TK6 cells were irradiated with various doses of gamma rays and incubated for 24 h in a modeled microgravity environment obtained by the Rotating Wall Vessel bioreactor. Cell survival, induction of apoptosis and cell cycle alteration were compared in cells irradiated and then incubated in 1g or modeled microgravity conditions. Modulation of genomic damage induced by ionizing radiation was evaluated on the basis of HPRT mutant frequency and the micronucleus assay. A significant reduction in apoptotic cells was observed in cells incubated in modeled microgravity after gamma irradiation compared with cells maintained in 1g. Moreover, in irradiated cells, fewer G2-phase cells were found in modeled microgravity than in 1g, whereas more G1-phase cells were observed in modeled microgravity than in 1g. Genomic damage induced by ionizing radiation, i.e. frequency of HPRT mutants and micronucleated cells, increased more in cultures incubated in modeled microgravity than in 1g. Our results indicate that modeled microgravity incubation after irradiation affects cell response to ionizing radiation, reducing the level of radiation-induced apoptosis. As a consequence, modeled microgravity increases the frequency of damaged cells that survive after irradiation.     

Space Microbiology

Summary: The responses of microorganisms (viruses, bacterial cells, bacterial and fungal spores, and lichens) to selected factors of space (microgravity, galactic cosmic radiation, solar UV radiation, and space vacuum) were determined in space and laboratory simulation experiments. In general, microorganisms tend to thrive in the space flight environment in terms of enhanced growth parameters and a demonstrated ability to proliferate in the presence of normally inhibitory levels of antibiotics. The mechanisms responsible for the observed biological responses, however, are not yet fully understood. A hypothesized interaction of microgravity with radiation-induced DNA repair processes was experimentally refuted. The survival of microorganisms in outer space was investigated to tackle questions on the upper boundary of the biosphere and on the likelihood of interplanetary transport of microorganisms. It was found that extraterrestrial solar UV radiation was the most deleterious factor of space. Among all organisms tested, only lichens (Rhizocarpon geographicum and Xanthoria elegans) maintained full viability after 2 weeks in outer space, whereas all other test systems were inactivated by orders of magnitude. Using optical filters and spores of Bacillus subtilis as a biological UV dosimeter, it was found that the current ozone layer reduces the biological effectiveness of solar UV by 3 orders of magnitude. If shielded against solar UV, spores of B. subtilis were capable of surviving in space for up to 6 years, especially if embedded in clay or meteorite powder (artificial meteorites). The data support the likelihood of interplanetary transfer of microorganisms within meteorites, the so-called lithopanspermia hypothesis.     

Effect of Microgravity on Fungistatic Activity of an α-Aminophosphonate Chitosan Derivative against Aspergillus niger

Biocontamination within the international space station is ever increasing mainly due to human activity. Control of microorganisms such as fungi and bacteria are important to maintain the well-being of the astronauts during long-term stay in space since the immune functions of astronauts are compromised under microgravity. For the first time control of the growth of an opportunistic pathogen, Aspergillus niger, under microgravity is studied in the presence of α-aminophosphonate chitosan. A low-shear modelled microgravity was used to mimic the conditions similar to space. The results indicated that the α-aminophosphonate chitosan inhibited the fungal growth significantly under microgravity. In addition, the inhibition mechanism of the modified chitosan was studied by UV-Visible spectroscopy and cyclic voltammetry. This work highlighted the role of a bio-based chitosan derivative to act as a disinfectant in space stations to remove fungal contaminants.     

Effect of simulated microgravity on the production of IL-12 by PBMCs

During space flight immunity is altered. This phenomenon is partly due to the microgravity condition itself. Our earlier space experiments (INTERFERON) indicated that microgravity has a significant effect at the cellular level. In our subsequent terrestrial studies we applied the Rotating Cell Culture System (RCCS) developed by NASA to mimick microgravity on ground. Previously we reported that human peripheral blood mononuclear cells (PBMCS) respond to simulated microgravity conditions with elevated tumor necrosis factor-alpha (TNF-alpha) production. We extended our investigations to the production of interleukin (IL)-12 under modelled microgravity conditions by separated PBMCs. In simulated microgravity we found significantly elevated level of secreted IL-12 compared to static, standard tissue culture conditions. Following a maximum of TNF-alpha production at 24 hours, the peak of IL-12 production was observed at 48 hours after the start of the experiment. Our results suggest that simulated microgravity favors the establishment of a Th1 type cytokine response.     

Synergy between stresses: an interaction between spaceflight‐associated conditions and the microgravity response

Gravity is the one constant, ubiquitous force that has shaped life on Earth over its 4.8 billion years of evolution. But the sheer inescapability of Earth’s gravitational pull has meant that its influence on Earth’s organisms is difficult to study. Neutralization of the gravity vector (so‐called simulated microgravity) by random movement in three‐dimensional space is the best option for Earth‐based experiments, with spaceflight alone offering the possibility to assess the effects of an extremely reduced gravitational field (microgravity). However, the technical constraints associated with spaceflight introduce complications that can compromise the interpretation of microgravity experiments. It can be unclear whether changes detected in these experiments reflect additional spaceflight‐related stresses (temperature shifts, vibrational effects, radiation exposure, and so on) as opposed to the loss of gravitational force per se. In this issue, Herranz et al. (2010) report a careful study in which the effects of simulated and actual microgravity on gene expression in Drosophila melanogaster were compared and the effects of the flight‐associated stresses on the microgravity responses were investigated. A striking finding emerged. The additional stresses associated with the spaceflight experiment altered the response to microgravity. Despite controlling for the effects of these stresses/constraints, the group found that responses to microgravity are much stronger in the stressed/constrained background than in its absence. This interaction of gravity with other environmental influences is a novel finding with important implications for microgravity research and other situations where multiple stress factors are combined.     

Analysis of cell survival after multiple fractions per day and low-dose-rate irradiation of two in vitro cultured rat tumor cell lines

Two rat tumor cell lines which differ significantly in radiosensitivity, a rhabdomyosarcoma (R-1) and a ureter carcinoma (RUC-2), were treated with multiple fractions per day and low-dose-rate gamma radiation. The purpose of these experiments was to investigate (i) the influence of fraction size and interfraction interval on repair of sublethal damage (SD) and (ii) whether low-dose-rate irradiation can be simulated by giving multiple fractions per day which might be applied in clinical treatments. In both cell lines, multiple doses were given at 1- to 4-hr intervals. SD repair was at a maximum in 2 hr but did not reach the theoretically expected level. For both cell lines, survival at higher total doses was different from that theoretically expected if repair of SD was assumed to be completed and at the maximum level. To account for the observation that less than complete repair of SD occurred, theoretical survival curves were calculated with the assumption of a constant but less than 100% level of SD repair. Experimental data correlated well with these calculated curves. There were only very small differences in survival after the different multiple fractions per day regimens. Survival after irradiation at a dose rate of 1.00 Gy/hr was found to be similar to that after multiple fractions per day.     

微重力环境影响植物生长发育的研究进展

微重力是最独特的空间环境条件之一,研究微重力对不同植物种类以及不同植物部位的影响是空间生物学的重要内容之一,对于建立生物再生式生命保障系统意义重大。生物再生式生命保障系统是未来开展长期载人空间活动的核心技术,其优势在于能在一个密闭的系统内持续再生氧气,水和食物等高等动物生活必需品,植物部件是生物再生式生命保障系统的重要组成部分。了解和掌握微重力对植物生长发育的影响,有助于采取有效的作业制度确保其正常生长发育和繁殖,是成功建立生物再生式生命保障系统的首要关键。该文就植物在空间探索中的地位和作用,地面模拟微重力的装置以及国内外有关微重力对植物的影响做一综述。现有的研究结果包括,未来长期的载人航天任务需要植物通过光合作用为生物再生式生命保障系统提供部分动物营养、洁净水以及清除系统中的固体废物和二氧化碳;三维随机回旋装置是目前地面上模拟微重力效应的主要装置之一,尤其适用于植物材料的长期模拟微重力处理;国内外有关微重力对植物影响的报道生理生化水平多集中在植物的生长发育和生理反应,比如表型变化或者与重力相关的激素或者钙离子的再分配,细胞或亚细胞水平主要有细胞壁、线粒体、叶绿体以及细胞骨架等,基因和蛋白质表达水平的研究对象主要为拟南芥。由于实验方法和材料之间的差异,微重力对不同植物或者植物不同部位在各个水平的影响效果并不一致,未来需要开展更多的相关研究工作。     

Recovery of Deinococcus radiodurans from radiation damage was enhanced under microgravity.

Effect of microgravity on recovery of bacterial cells from radiation damage was examined on the IML-2 mission in 1994 using extremely radioresistant bacterium Deinococcus radiodurans. The cells were lyophilized and exposed to 60Co gamma-rays with doses 2 to 12 kGy before the space flight. At the end of the mission, the cells were mixed on board with liquid nutrient medium to allow the cells to start recovery process from the radiation damage. Afterwards the cells were stored at 4 degrees C until landing. The influence of cosmic radiation was negligible, because total absorbed dose of space radiation measured during the mission was less than 2 mGy and this bacterium does not decrease its viability after both gamma-rays and high-LET heavy charged particles irradiation with doses up to 5 kGy. The survival of the cells incubated in space increased significantly compared with the ground controls, suggesting that the recovery of this bacterium from radiation damage was enhanced under microgravity.     

Effect of simulated microgravity on human lymphocytes

During space flight the function of the immune system changes significantly. Several papers reported that postflight the number and the proportion of circulating leukocytes in astronauts are modified (Leach, 1992), the in vitro mitogen induced T cell activation is depressed (Cogoli et al., 1985; Konstantinova et al. 1993) and there are detectable differences in cytokine production of leukocytes as well (Talas et al. 1983; Batkai et al. 1988; Chapes et al. 1992). One of the possible modifying forces is the microgravity condition itself. Our aim was to analyse mechanisms responsible for changing leukocyte functions in low gravity environment. For terrestrial simulation of microgravity we used a Rotary Cell Culture System (RCCS) developed by NASA. We investigated the effect of simulated microgravity on separated human peripheral blood mononuclear cells (PBMCs). We detected the populations of different cells by antibodies conjugated to fluorofors using a Flow Cytometer. Since space flight reduces the number of peripheral blood lymphocytes (Stowe et al., 1999) we supposed that apoptotic (programmed cell death) processes might be involved. This hypothesis was supported by the result of our earlier experiment demonstrating that simulated microgravity increased the level of secreted Tumor Necrosis Factor-alpha (TNFalpha, a known apoptotic signal molecule) significantly (Batkai et al. 1999).     

Cell interactions in microgravity: cytotoxic effects of natural killer cells in vitro

To study cell-to-cell interactions in microgravity we examined the functional activity of natural killer cells on board of the ISS. NK cells are the effector cells with direct cytotoxic activity to oncogenic, virus-infected cells and cells with modified differentiation. Ground-based experiments have shown that the examination of target cell lysis after incubation with NK cells is a simple and informative model for studying the influence of microgravity. NK cytotoxicity was measured as the value of non-degradeted labeled myeloblasts (K-562) after 24 hrs exposure with human lymphocytes in suspension. A special device was developed for space flight experiments. Human cultured lymphocytes and labeled K-562 cells were loaded into special syringes and delivered to the Russian segment of the ISS. Cosmonauts prepared co-cultured suspensions during the first day of microgravity, exposed them at 37 degrees C for 24 hrs and then separated H3-labeled cells on special filters. The results of ISS-8 mission showed that human NK cells in vitro remain lysis activity toward target cells in microgravity. The basal level of NK cytotoxicity was low and we did not found significant differences between "control" and "flight" values. Interferon production during the interaction between immune and target cells (ratio 10:1) in microgravity did not differ compared with ground-based control experiments. Ground exposure of the same lymphocyte samples with K-562 cells to 24 hrs clinorotation also did not lead to significant differences. These experiments paved the way for understanding the cell interaction mechanisms in space flight and the obtained results suggest that microgravity does not disrupt the interaction of NK cells with tumor cells.     

Leukocyte Activity Is Altered in a Ground Based Murine Model of Microgravity and Proton Radiation Exposure

Immune system adaptation during spaceflight is a concern in space medicine. Decreased circulating leukocytes observed during and after space flight infer suppressed immune responses and susceptibility to infection. The microgravity aspect of the space environment has been simulated on Earth to study adverse biological effects in astronauts. In this report, the hindlimb unloading (HU) model was employed to investigate the combined effects of solar particle event-like proton radiation and simulated microgravity on immune cell parameters including lymphocyte subtype populations and activity. Lymphocytes are a type of white blood cell critical for adaptive immune responses and T lymphocytes are regulators of cell-mediated immunity, controlling the entire immune response. Mice were suspended prior to and after proton radiation exposure (2 Gy dose) and total leukocyte numbers and splenic lymphocyte functionality were evaluated on days 4 or 21 after combined HU and radiation exposure. Total white blood cell (WBC), lymphocyte, neutrophil, and monocyte counts are reduced by approximately 65%, 70%, 55%, and 70%, respectively, compared to the non-treated control group at 4 days after combined exposure. Splenic lymphocyte subpopulations are altered at both time points investigated. At 21 days post-exposure to combined HU and proton radiation, T cell activation and proliferation were assessed in isolated lymphocytes. Cell surface expression of the Early Activation Marker, CD69, is decreased by 30% in the combined treatment group, compared to the non-treated control group and cell proliferation was suppressed by approximately 50%, compared to the non-treated control group. These findings reveal that the combined stressors (HU and proton radiation exposure) result in decreased leukocyte numbers and function, which could contribute to immune system dysfunction in crew members. This investigation is one of the first to report on combined proton radiation and simulated microgravity effects on hematopoietic, specifically immune cells.