Influence of factors of short-term suborbital flight on functional activity of human lymphocytes
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1
Federal Medical & Biological Agency of Russia, Russia
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2
Research Institute of General Pathology and Pathophysiology, the Russian Academy of Medical Sciences, Russia
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3
Skolkovo Institute of Science and Technology, Russia
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4
Moscow Polytech, Russia
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5
Central Aerological Observatory, Federal State Budget Institution, Russia
Many different technologies and methods are used for the evaluation of the influence of spaceflight factors on the body. For instance, the effects of microgravity are studied in vitro on a clinostat (for cells) and in vivo using the tail suspension test (for mice). The effects of radiation can be easily modeled under in ground conditions. Exposure to spaceflight factors, even during short-term missions, may affect the number and functions of studied cells. Any extreme exposure triggers activation of general adaptation systems, but the rate and degree of these processes and, most importantly, the degree of exhaustion and the development of compensatory responses can vary. These factors ultimately determine viability of the organism.
Despite of the fact that the main trend in international astronautics is the transition from near-Earth orbital flights to long-distance spaceflights to the Moon and Mars, the initial phase of space tourism will involve primarily low-earth suborbital trajectories. Suborbital crew and passengers will be exposed to various potentially dangerous factors during flights. The main problem in evaluation of biological risk of anthropogenic and other influences in suborbital flights is the necessity to analyze the effect of doses not surpassing or slightly surpassing the threshold doses. In this range the response to the studied factors is maximally individual. Biological consequences of these influences can be revealed only in multilevel studies. High radiation level in combination with other stress factors can induce damage to cell structures, changes in their functioning, which, in turn, can lead to activation of defense systems. Taking into account the fact that increased radiation level is related to solar activity and depends on flight altitude, even-short-term exposure to extreme factors may lead to exhaustion of the adaptive resources and under these conditions hypersensitivity of the organism can develop even after subthreshold exposures.
An established space tourism industry will involve longer orbital flights and possibly permanent facilities including hotel and resort facilities. These flights and facilities present an additional risk because they involve longer durations of potential exposure to elevated radiation levels [1].
The main goal of this experiment was to investigate the cellular and molecular responses of human blood cells (lymphocytes) to gravity changes.
Methods. On July, 27 2017 meteorological rocket MMR-06 was launched. It carried a special thermostat (370C) module with two 75-ml cultural flacks. Start acceleration was ~40 g. The measurement phase covered the altitude range of 64431 m. Ballistic flight to the apogee took 116 sec, microgravity part of еру flight lasted 60 sec. The horizontal distance of the flown part was about 30 km.
Whole blood lymphocytes from a healthy female donor were routinely isolated by centrifugation in Diacoll-1077 density gradient. The medium was centrifuged (100,000g, 2 h) and filtered (0.2-µm Millipore filter) before the experiment to avoid extra particles during dynamic light scattering analysis. After washout, the cells in RPMI-1640 medium were transferred to 2 flight and 2 ground flasks in a concentration 0.44x106 cells/ml. Flasks temperature during the flight was 38+100C, the ground flasks were kept at the same temperature.
After landing the flight and ground cell suspensions were centrifuged (3500 rpm, 15 min). The cells were used for comet assay, culture fluids were frozen.
Electromagnetic radiation is among flight factors producing maximum damaging effect on cell DNA. Control irradiation of samples was performed at the Central Aerological Observatory. The electromagnetic radiation from the equipment inside the rocket head ("upper" sample), the maximum theoretical irradiation from the equipment inside the rocket head ("side" sample), and the effect from the tracking radar ("radar" sample) were simulated. The exposure for the first two samples was 2 h and for radar sample - 30 min.
Subfractional composition of the culture fluid was determined using laser correlation spectrometer for dynamic light scattering. The method is based on a change in spectral characteristics of monochromatic coherent radiation of a helium-neon laser. Measurement of the culture fluid (0.2 mL) and processing of the spectra were performed in accordance with the conventional technique [2,3]. The particle size distribution presented after mathematical processing in the form of a histogram allows characterization of dispersion composition of a certain biological fluid and classification of the distribution according to chosen informative zones of the spectrum.
The Comet Assay or single cell gel electrophoresis (SCGE) assay is a quick, sensitive, and relatively simple method for detecting DNA damage at the level of individual cells. Analysis of DNA damage degree was performed in accordance with the protocol [4].
Results
Percent of live cells tested with eosin was almost the same in both flight and ground samples – 91 and 92%, respectively. Control ground experiments were performed to reveal separate effects of some flight factors – temperature, acceleration, electromagnetic field, etc. Temperature variations and overload 40 g produced no changes in the genetic material. Comet test revealed difference in degree of cell DNA damage. Cells after flight demonstrated mainly pronounced degree of DNA damage (Fig.1).
Fig.1 The percentage of comets of different types in the samples
Taking into account the equal quantity of normal cells in both samples, we suppose that the influence of flight factors consists in accelerated destruction of cells with preexisting defects.
DNA comet analysis showed the maximum percentage of cells with damaged DNA in the radar sample.
The presence of a large number of type 1 DNA comets in this point indicates massive de novo injuries caused by irradiation.
Dynamic light scattering of cultural fluids showed shifts in their composition. Flight samples contained more 67-123-nm particles than control ones with a maximum at 91 nm. This may indicate that DNA fragments are released from cells to the culture fluid (Fig.2).
Fig.2 LC histograms of culture fluid samples .
The difference between the LC histograms of the test sample and ones characterizing the medium after different irradiation regimens shows that electromagnetic radiation induced changes in the "radar" and "side" samples similar to the changes observed during the experiment (namely, increase in the percentage contribution of 67-123-nm particles into the light scattering).
Conclusions
Factors of suborbital flight did not cause additional cell death, but aggravated destruction processes in the damaged cells.
Electromagnetic irradiation in points "radar" and "side" is considered by us as the theoretically maximum possible exposure, however, similar shifts in LC histograms showed considerable contribution of the effect of electromagnetic radiation into complex effect of all flight factors.
Acknowledgements
Authors would like to express their gratitude to the Resource Center of Kurchatov complex NBIKS NCI Technologies, National research Center "Kurchatov Institute" (Moscow, Russia) for kindly support in gel documenting and image processing.
References
1. Bevelacqua, J. J. (2017). Radiation protection consequences of the emerging space tourism industry. JJ Earth Science, 1, 1-11.
2. Karganov, M., Alchinova, I., Arkhipova, E., & Skalny, A. V. (2012). Laser correlation spectroscopy: nutritional, ecological and toxic aspects. In Biophysics. InTech.
3. Karganov, M., Sychev, V., Shenkman, B. S., Arkhipova, E., Alchinova, I., & Medvedeva, U. (2014). Differentiation of changes in serum homeostasis of laboratory animals after spaceflight on board of the BION-M1 satellite. In 40th COSPAR Scientific Assembly (Vol. 40).
4. http://www.cometassay.com
Keywords:
lymphocyte,
Suborbital flight,
Gravity changes,
Cell Death,
Electromagnetic Radiation
Conference:
39th ISGP Meeting & ESA Life Sciences Meeting, Noordwijk, Netherlands, 18 Jun - 22 Jun, 2018.
Presentation Type:
Extended abstract
Topic:
Immune System and Radiations
Citation:
Alchinova
I,
Polyakova
M,
Karganov
M,
Baranov
M,
Mullin
N,
Kalinkin
S,
Morozov
K,
Balugin
N and
Yushkov
V
(2019). Influence of factors of short-term suborbital flight on functional activity of human lymphocytes.
Front. Physiol.
Conference Abstract:
39th ISGP Meeting & ESA Life Sciences Meeting.
doi: 10.3389/conf.fphys.2018.26.00018
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Received:
02 Dec 2018;
Published Online:
16 Jan 2019.
*
Correspondence:
Dr. Irina Alchinova, Federal Medical & Biological Agency of Russia, Moscow, Moscow Oblast, Russia, alchinovairina@yandex.ru