The Effect of Long-term Space Flights on Human Urine Proteins Functionally Related to Endothelium

It is known that long-term space flights lead to dysregulation of the cardiovascular system, and the endothelium is the most important functional element of such dysregulation. In order to find the signs of endothelial dysfunction in cosmonauts who have been in long-term space flights, we collected urine samples from 21 cosmonauts before the flight and on the first and seventh days after landing. The urine samples were investigated by chromatography–mass spectrometry analysis. Proteins were identified using the MaxQuant software and the SwissProt database. The software package Perseus was used for semi-quantitative analysis. The reconstruction of associative molecular networks was performed using the ANDSystem software. We identified 200 different proteins in urine samples of 21 Russian cosmonauts. The ANDSystem software made it possible to determine seven processes related to endothelium functioning. These processes had direct relations to 17 urine proteins, which were functionally associated with the endothelium. At the same time, eight proteins (such as serotransferrin, prostate-specific antigen, fibrinogen gamma chain, UFO tyrosine kinase receptor, aminopeptidase N, vascular cell adhesion molecule 1, osteopontin, and syndecan-4) were significantly changed (p < 0.01) at different points of the recovery period (the first and seventh days). Thus, we performed the first study of the urine protein composition in cosmonauts for the evaluation of signs of endothelial dysfunction after space flight using proteomics methods.     

Detection of Renal Tissue and Urinary Tract Proteins in the Human Urine after Space Flight

The urine protein composition samples of ten Russian cosmonauts (male, aged of 35 up to 51) performed long flight missions and varied from 169 up to 199 days on the International Space Station (ISS) were analyzed. As a control group, urine samples of six back-up cosmonauts were analyzed. We used proteomic techniques to obtain data and contemporary bioinformatics approaches to perform the analysis. From the total number of identified proteins (238) in our data set, 129 were associated with a known tissue origin. Preflight samples contained 92 tissue-specific proteins, samples obtained on Day 1 after landing had 90 such proteins, while Day 7 samples offered 95 tissue-specific proteins. Analysis showed that consistently present proteins in urine (under physiological conditions and after space flight) are cubilin, epidermal growth factor, kallikrein-1, kininogen-1, megalin, osteopontin, vitamin K-dependent protein Z, uromodulin. Variably present proteins consists of: Na(+)/K(+) ATPase subunit gamma, β-defensin-1, dipeptidyl peptidase 4, maltasa-glucoamilasa, cadherin-like protein, neutral endopeptidase and vascular cell adhesion protein 1. And only three renal proteins were related to the space flight factors. They were not found in the pre-flight samples and in the back-up cosmonaut urine, but were found in the urine samples after space flight: AFAM (afamin), AMPE (aminopeptidase A) and AQP2 (aquaporin-2). This data related with physiological readaptation of water-salt balance. The proteomic analysis of urine samples in different phases of space missions with bioinformation approach to protein identification provides new data relative to biomechemical mechanism of kidney functioning after space flight.     

Antibodies to muscle fiber antigens in the cosmonauts after a space flight]

The study of serum samples, obtained from 15 cosmonauts before and after space flights, with the use of the indirect fluorescent method showed that in 7 cosmonauts antibodies to different elements of the human heart muscle appeared after flights. Strong and very strong luminescence of the elements of heart muscle tissue was detected in the cosmonauts after the third space flight. When studying the sera on sections of bovine heart muscle tissue, the reactions of the sera taken before and after flights were found to have no essential differences.     

Calcium metabolism and its regulation in man during adaptation to microgravitation

This work generalizes the results of studies of calcium metabolism in the participants of long-term space flights of 30 to 438 days on the Salyut and Mir orbital stations during 1978–1998. The results of pre- and postflight examination of 44 cosmonauts (18 subjects participated twice in long-term space flights) were analyzed. After space flights of medium (of 3 to 6 months) and long (of 6 to 14 months) duration, the total blood calcium content was increased, mainly due to its ionized fraction; the blood level of parathyroid hormone was significantly increased and the level of calcitonin was decreased. The content of osteocalcin was increased after space flights. Calcium kinetics was studied using stable isotopes in three cosmonauts before, during, and after the 115-day flight. During the flight, intestinal absorption of calcium and its gastrointestinal excretion were decreased, whereas its renal excretion was increased. Early postflight intestinal absorption was, on average, lower than during the flight, whereas intestinal excretion increased. Both renal and intestinal excretion of calcium were not normalized 3.5 to 4.5 months after the glight. The mathematical models used for evaluating the rates of main calcium flows revealed increased bone tissue resorption that resulted in the negative bone balance during the flight. The conclusion about the decreased rate of bone tissue remodeling and its increased resorption was confirmed by biochemical data, including endocrine markers.     

Animals and spaceflight: from survival to understanding

Animals have been a critical component of the spaceflight program since its inception. The Russians orbited a dog one month after the Sputnik satellite was launched. The dog mission spurred U.S. interest in animal flights. The animal missions proved that individuals aboard a spacecraft not only could survive, but also could carry out tasks during launch, near-weightlessness, and re-entry; humans were launched into space only after the early animal flights demonstrated that spaceflight was safe and survivable. After these humble beginnings when animals preceded humans in space as pioneers, a dynamic research program was begun using animals as human surrogates aboard manned and unmanned space platforms to understand how the unique environment of space alters life. In this review article, the following questions have been addressed: How did animal research in space evolve? What happened to animal development when gravity decreased? How have animal experiments in space contributed to our understanding of musculoskeletal changes and fracture repair during exposure to reduced gravity?     

Capillary filtration rate, venous compliance, and blood flow in arms and legs do not change during bed rest for 20 days

It has been generally accepted that pooling of the blood in the legs is one reason for the orthostatic intolerance experienced after space flights. This is also the reasoning behind the application of anti-G suits during reentry after space flights. Fighter pilots also use the anti-G suit, the hypothesis being that this prevents the pooling of blood in the legs. In order to investigate if immobilization during bed rest would induce peripheral cardiovascular deconditioning we measured capillary filtration rate, venous compliance, and blood flow in arms and legs during bed rest.     

The volume of extracellular fluid under conditions of long-term space flights

The volume of extracellular fluid (the bromine space) was determined in 18 cosmonauts 30 days before the start of a space flight and on the first day after landing. The duration of space flights on the Mir orbital station was from 126 to 438 days. Moreover, the volume of extracellular fluid was determined in seven cosmonauts directly during long-term space flights approximately two weeks before landing. After long-term space flights, the volume of extracellular fluid was decreased in all cosmonauts studied. The bromine space volume was significantly decreased compared to its initial preflight value. A decrease in the volume of extracellular fluid was caused not only by the reduction in the dense mass of the body but also by its dehydration. These processes developed independently of the duration of weightlessness but are mainly determined by the individual features of human beings.     

Effect of space flight factors on health of cosmonauts in the near and late term after space flights

Cytogenetical studies of cosmonauts' peripheral blood lymphocytes after space flights on MIR orbital station showed a statistically significant increase in the yields of radiation-induced chromosomal aberrations. However, similar studies with in vitro irradiation of biological objects with accelerated charged particles are of great importance for elucidation of the nature of cytogenetical damage induced in vivo. It is also important to investigate the structure of cosmonatus' diseases over their life, in particular, lens opacities and oncological diseases. Thus, the purpose of the investigations planned is to study cytogenetical damage in blood lymphocytes from cosmonauts after space flights on the ISS in vivo, as well as in donor blood lymphocytes after in vitro exposure to accelerated charged particles. The tasks of the project are as follows: determination of the yields and types of chromosomal aberrations in cosmonauts' blood lymphocytes before and after space flights, comparative studies of biological effects induced in vitro by different types of ionizing radiation in human blood lymphocytes in ground experiments, assessment of cytogenetical risks, analysis of the structure of cosmonatus' diseases comparing with that of whole population, study of the mortality and frequency of cataracts and oncological diseases in cosmonauts. The results to be obtained will be used for setting of health norms applied to the influence of radiations of different types, and for elaboration of measures to reduce health risks from space flight factors.     

Analysis of possible causes of activation of gastric and the pancreatic excretory and incretory function after completion of space flight at the international space station

A comparative analysis of the excretory and incretory activity of the stomach and pancreas in astronauts soon after completion of space flights of various durations was performed. An increase in the fasting activity of gastric and pancreatic enzymes and hormones (insulin and C-peptide) in blood, reflecting the increased excretory and incretory activity of organs of the gastroduodenal region developing in microgravity, was demonstrated. The absence of subjects infected with Helicobacter pylori in the space flight crew excluded the involvement of this microorganism in the mechanism underlying the increase in the gastric secretory activity. The absence of correlation between the increase in the secretory activity of organs of the gastroduodenal region and the duration of the space flight allowed us to rule out the hypokinetic mechanism, which is associated with the duration of exposure to microgravity. It was concluded that the main mechanism underlying the changes in the functional state of the digestive system in space flight may be determined by the rearrangement of the venous hemodynamics of organs of the abdominal cavity, unrelated to the duration of exposure to microgravity. It was shown that, after completion of space flights and in ground-based experiments simulating the hemodynamic rearrangement occurring in microgravity, the increase in the basal excretory activity of gastroduodenal organs was not caused by gastrin secretion and occurred simultaneously with an increase in the secretion of insulin, which is considered as a putative hormonal component of the hemodynamic mechanism.     

Cytogenetic characteristic of osteogenic cells in vitro as perspective predictors of osteopenia under microgravity

Mechanical stimulation of bone tissue determined by earth gravity is one of the main factors mediating the nature, rate and direction of functional adaptation of the bone system in the process of onto- and phylogenesis. Theoretically expected losses of bone mass under condition of mechanical load deficit under microgravity (osteopenia, osteoporosis) may become a factor that limits the duration of space flights. As a result of long-term studies some properties and regularities of change in human tissue after prolonged space flights (for 5-7 months) were established.     

Calcium metabolism characteristics in microgravity]

The results of research of calcium exchange parameters at cosmonauts taken part in long space flights (SF) onboard of orbital stations "SALUT" and "MIR" within 1978-1998 were generalized. The analysis of data received during observation of 44 cosmonauts (18 of them have taken part in long SF twice) was done. The observation was carried out before and after SF by duration 30-438 days. The content of a total calcium in blood serum was increased basically by the increase of its ionized fraction after flights of moderate (3-6 months) and large duration (6-14 months) along with the significant increase of PTH and decrease of calcitonin levels. The content of osteocalcin after SF was increased. Three cosmonauts participated in research of calcium kinetics using stable isotopes before, in time and after a 115-day SF. Reduction of intestinal absorption, excretion through a gastrointestinal tract, and increase of calcium excretion with urine were marked in time of SF. In early postflight period a level of intestinal absorption, on the average, was much lower than in SF, and the calcium removal through intestine was increased. Both renal and intestinal excretion of calcium were not normalized in 3.5-4.5 months after end of SF. Increase of resorbtive processes in bone tissues which induced negative bone balance during flight was observed in all test subjects, proceeding from estimations of speed of the basic calcium flows made on the basis of mathematical modeling. The conclusion about decrease in speed of bone tissue remodeling and strengthening of its resorption proves to be true by data of research of biochemical and endocrine markers.     

Simulated microgravity induce glutathione antioxidant pathwayin Xenopus laevis embryos

Space flights cause a number of patho-physiological changes. Oxidative damage has been demonstrated in astronauts after space flights. Oxidative stress is due to an imbalance between production of oxidant and antioxidative defence. In embryos of Xenopus laevis, the glutathione system is an inducible antioxidant defence. For this reason, we investigated the effect of gravity deprivation on endogenous antioxidant enzymes in X. laevis embryos developed for 6 days in a Random Positioning Machine. The results show that glutathione content and the activity of antioxidant enzymes increase in RPM embryos, suggesting the presence of a protective mechanism. An induction of antioxidant defence might play an important role for animals to adapt to micro-gravitational stress, possibly during actual space flights.     

Cytogenetic studies of blood lymphocytes of cosmonauts after long-ter, space flights

An analysis was performed of unstable chromosomal aberrations in peripheral blood of 36 cosmonauts after long-term space missions on "Mir" orbital station. 25 cosmonauts were examined before their flights to score spontaneous yields of cytogenetical damage. In all cases the doses absorbed by crews during space flights did not exceed permissible levels of irradiation, adopted for cosmonauts. The frequencies of chromosomal-type aberrations after space missions were found to increase significantly compared to the pre-flight levels. The yields of dicentrics and centric rings on the average were as high as 0.12 +/- 0.02 and 0.47 +/- 0.06% before and after the 1st flight, 0.18 +/- 0.05 and 0.71 +/- 0.11% before and after the 2nd flight respectively. During the inter-flight periods, usually lasted 1.5-2 years, the yields of chromosome damage lowered, but did not reach their spontaneous values. After each next flight the yields of chromosome aberrations increased again. The cytogenetical damage detected in cosmonauts' peripheral blood lymphocytes after chronic action of low doses of space radiation points out a possible increase in risks of stochastic effects in distant future for crews after long-term space missions.     

Quantitative target proteomics of chromosome 13 human blood plasma proteins

Quantitative proteomic analysis of 50 blood plasma samples of healthy volunteers who underwent a comprehensive medical examination and were found eligible for space flights was performed. As a result of directed mass spectrometric analysis, signals for 128 proteins, which accounted for nearly 40% of the total number of chromosome 13 gene products, were detected. The analysis of interindividual variation of concentrations of chromosome 13 proteins showed the presence of a pool comprising 41 proteins with a low variation (CV < 30%), which can potentially be used as biomarkers.     

Endocrine responses to space flights

Simultaneously with human space flights several series of observations were performed by using experimental animals--mainly rats--exposed to space flights on board of special satellites BION-COSMOS or in Shuttle Transportation Systems (STS). The aims of these experiments were to study in more details: the mechanisms of the changes in bones and skeletal muscle, the alterations of the function of immune system, the radiation effects on organism, the mechanism of the changes of endocrine functions, the evaluation of the role of hormones in alteration of metabolic processes in organism. The advantages of these animal experiments were the possibilities to analyze not only the plasma samples, but it was possible to obtain samples of organs or tissues: for morphological and biochemical analysis for studies of the changes in enzyme activities and in gene expressions, for measurement of metabolic processes and for investigation of the hormone production in endocrine glands and estimation of the response of tissues to hormones. It was also possible to compare the endocrine response to spaceflight and to other stress stimuli. These animal studies are interesting for verification of some hypothesis in the mechanism of adaptation of human organism to the changes of gravity. The disadvantage was, however, that the animals in almost all experiments could be examined only after space flight. The actual inflight changes were investigated only in two SLS flights. In this short review it is not possible to evaluate all hormonal data available on the response of endocrine system to the conditions of space flights. Therefore we will concentrate on the response of pituitary adrenocortical system, pituitary thyroid and pituitary gonadal functions.     

卫星搭载对甘草种子萌发中生理特性的影响

为探讨卫星搭载处理对甘草种子萌发中生理特性的影响,本文运用卫星搭载技术对甘草种子进行太空诱变处理,考察了卫星搭载后甘草种子（卫星1号,GF）在干旱胁迫条件下,萌发过程中的发芽率、胚根长势、可溶性蛋白变化以及过氧化物酶和过氧化氢酶的活力,并与地面对照组种子（地面组,GN）进行了比较。结果表明,在干旱胁迫条件下（水势－1．0MPa）,飞行组种子的发芽率、胚根长势要高于地面对照组,其萌发过程中的过氧化物酶、过氧化氢酶活力也高于地面对照组。卫星搭载处理后,甘草种子的抗旱性能增强。     

The influence of space flights on water-electrolytes turnover and its regulation

A study of water-electrolyte exchange, the condition of water milieu of the organism, and the volume- and electrolyte homeostasis regulation in space flights, and also in postflight period has shown the important role of the water-salt homeostasis in adaptation of the human and animal organism to weightlessness. Obviously, downturn of food consumption, renal excretion and the intestine output seem to be caused by suppression of activity of mechanisms of ion deposition. The most intensive changes of the liquid milieu volumes occur in the first days of weightlessness or in its ground simulation. And, with prolonged duration, the changes of extracellular liquid volume and the volume of plasma do not extend. After termination of long space flights, activation of renin-aldosterone systems occurs as well as a decrease in efficiency of antidiuretic hormone, misbalance of pressor/unpressor prostanoids. In the period of re-adaptation after space flights, development of desensitization of kidneys to endogenous ADH occurs. This is the basis for researches directed to improvement of the existing scheme of correction of the hydrogenous status of the astronaut organism in the closing stage of flight.     

Electrophoretic and immunochemical research of rat urine proteins in dynamics after intravenous injection of thorium dioxide (thorotrast)

Rats were treated with a single intravenous injection of thorotrast (thorium dioxide)--the source of alpha-rays. Dynamic investigation of urine protens of rats by methods of electrophoresis and immunoelectrophoresis was carried out during 22 months after thorotrast injection. Already the month after drug injection the selectivity of tubular reabsorbtion was disturbed. Three months after thorotrast injection the content of urinal proteins of tissue (in particular renal) origin was decreased. Finally the selectivity of renal filtration of proteins was damaged 4-6 months after thorotrast introduction. Serum proteins which were absent in normal urine (for example transferrin and lipoproteins) appeared in urine of affected rats. The urine proteins of serum origin were less degraded than those in normal urine. The alterations of glomerular filtration was increased up to 20-22 months when the spectrum of urine proteins became similar to the spectrum of serum proteins. The death of treated rats was occurred in this period. Thus the monitoring of urine proteins of rats treated with alpha-ray producing preparation throtrast allows to register the successive alterations of reabsorbtion, excretion and filtration functions of kidney.     

Structural and metabolic characteristics of human soleus fibers after long duration spaceflight

The fiber size decline, alterations in fiber metabolic potential and increase of connective tissue component were shown in human m. vastus lateralis after short and long-duration space flights and in m.soleus and m.vastus lateralis after 120 day head down tilt bed rest. It is known from rat and monkey studies that the exposure to weightlessness leads to the most pronounced changes in postural muscles, e.g. m.soleus. It was shown that 17 day space flight induced significant decrease of fiber cross-sectional area and slow-to-fast fiber type transformation in human soleus. But in the cited work the fiber population under study was limited like in most single fiber technique analyses. The present study was purposed to investigate the structural and metabolic properties of soleus muscle in Russian cosmonauts exposed to 129-day space flight on board of the International Space Station.     

A policy of anticipation as a strategic objective of space biology and medicine at the present stage

This article reflects the ongoing debates in Russia regarding paths of innovative development and the role that fundamental science has played in the development of technology critical for national security and for its breakthrough potential. Alternative routes of technical development include variations in priority support for those points of growth, in which Russia has attained and steadily held the leading position, occupying a prominent place in the international division of labor. Russia’s space program is a good example of the successful implementation of a national program and provides a demonstration of the country’s leadership role in this area of human activities. This article presents an analysis of the factors and circumstances in Russia that predetermined, in the early, “Gagarin” period of piloted space flights, its winning of the leading position. They also determined the vector of the development of manned space flight for many years to come. Even taking into account the host of issues with the implementation of the International Space Station (ISS) utilization program and the planning of manned flights to the Moon and Mars, the unique experience of preparations and the conducting of research and tests with humans in space—the enormous groundwork in fundamental biomedical research over the past 50 years of piloted flights—provides the basis for an optimistic prognosis for gaining headway with essentially new, ambitious space projects. The key question is whether the proactive strategy of prioritized development and the affirmation of the role of manned space flights as the most integrated and science-intensive sector of innovative achievement will be realized. The development of the space industry depends upon the answer, not only at the present stage but in the long-term as well, as does the fate of the national fundamental space sciences, an integral part of which is space biomedicine.