It is known that some of the surviving cells after irradiation can produce functionally modified descendants, who have for many generations with a high frequency occur de novo chromosome aberrations and predominantly point mutations, in certain cases increase cell death by apoptosis [Little J. B. 2000, Seymour C. B., Mothersill C. & Alper T. 1986, Baverstock K. 2000]. The accumulation of such mutations suggests a high probability disorders in the bases caused by oxidative stress [Little D. B. 2007]. It is known that even in non-irradiated cells residing in close proximity to irradiated cells, induction of chromosomal instability is possible.

Placing of non-irradiated cells (NirC) in the culture medium of irradiated cells (IrC), soon leads to the appearance in the descendants of (NirC) of chromatid and chromosome aberrations, micronuclei, gene mutations that increase the content of the transformed cells

[Pfeiffer P., Gottlich N. & Reichenberger S. 1996, Seymour C. B., Mothersill C. 1997, Johansen C. O. 1999]. The phenomenon of transfer of an altered state from the modified cells under damage factors to unmodified cells was named "bystander effect" (BSE). It was first described in Chinese hamster [Nagasawa H, Little J. B. 1992] cells and was later found in different types of cells after exposure to damaging factors of different nature [Azzam E. I., Little J. B. 2003, Azzam, E. I., de Toldeo, S. M. & Little, J. B. 2003, Chakraborty A, Held K. D. & Prise K. 2009, Mothersill, C & Seymour, C. 2001]. Thus, cells that have not been laid through the tracks of IR are "bystanders" of radiation injuries caused by other irradiated cells. Reactive oxygen species (ROS) play an important role in the mechanisms of signal transmission to "bystander" cell [Kudryashov Yu. B. 2004]. Reinforced ROS production in NirC incubated in medium with serum, irradiated a-particles, or with supernatant of the suspension of IrC [Narayanan P. K., Googwin E. YH, Lehnert B. E. 1997] or after contact by IrC [Grosovsky A. J. 1999] is shown.

Irradiated cells produce several "bystander" signals — cytokines, fragments of DNA (from apoptotic cells) or other factors of protein nature. These factors cause a change in oxidative metabolism and gene expression profiles in IrC, and induce enhanced production of highly ROS [Watson G. E., Lorimore S. A., Macdonald D. A., 2000, Ermakov A. V., Kon’kova M. S., Kostyuk S. V. 2009, Snyder A. R. 2004, Lorimore S. A., Coates P. J., Scobie G. E. 2001]. In addition to chemical modification of DNA nucleotides the formation of radicals can also lead to changes in the higher levels of organization structure of the molecule to the secondary, tertiary and quaternary conditions [Nobler М. Р. 1969]. Therefore the level of oxygen and antioxidants influence the quantitative yield and quality of damaged bases of DNA in IrC.

Signal transmission of such lesions through the culture medium is typical for BSE induced by IR from low linear energy transfer [Mothersill C., Seymour C. B. 1998]. In our study we used a hypothesis about the ability of y-rays to generate in a organism of mammals part of "bystander" signals that can be distributed in the environment of the body and affect distantly unexposed tissue. At the same time we take as an axiom that some of these of signals has a free radical nature, in particular, can be represented by the ROS. Scheme of the origin and development of BSE corresponding to this hypothesis is shown in Figure 18.

We used a biological model, which allows you to multiply the amount of cellular material to strengthen the "bystander" signals in the intercellular space. For this purpose females of mice Balb/c and C57bl/6 were irradiated. It is known that mice C57Bl/6 is moderately sensitive to ionizing radiation, LD50/30 = 6.70 Sv, and Balb/c line of mice is highly sensitive to IR, LD50/30 = 5.85 Sv [Blandova Z. K., Dushkin V. A. & Malashenko A. N. 1983, Storer J. B 1966]. After exposure to у-fields from organs and tissues of the mice (peripheral blood, spleen, liver, bone marrow and brain — astroglia) cell suspensions were obtained.

Protocol modes of mice irradiation:

1. Total one-time 16-hour exposure y-radiation from small samples of nuclear fuel IV unit of ChNPP modified during the accident in 1986, which were evenly distributed under the cages with the exhibited animals (30 mice per line) and formed a fairly uniform horizontal y — field with the exposure dose rate of about 8.7 • 10-4 Gy sec, allowed to reach a total dose of about 5.0 Sv (85% of the LD50/30).

2. The total external y-radiation exposure from a specially constructed flat concrete bars, containing soil from the Red Forest with a specific activity of 30 kBq/kg and creating exposure power ~ 52.2 mcSv/h. They were placed under cages with animal at 231 days. Total external dose was about 0.29 Sv. Radioactive soil we previously burned for the destruction of organic compounds.

3. Long-term (over 74 days) incorporation of 137Cs with a drink (at the rate of 6.0 kBq per mouse per day) led to accumulation radioactivity from 14 to 24 kBq in the mouse body, which was identified in the y-spectrometer CP-4900V (Nokia, Finland). The average total estimated activity was 17.0 ± 1.0 kBq per animal.

Identification model of "bystander" signals

Replacing the living environment of mice

Non-irradiated and irradiated cells obtained from organs and tissues of mice kept in a nutrient medium RPMI-1640 supplemented with 5% syngenic serum for 3 hours, after that the NirC placed in culture medium of IrC (Figure 19)

Fig. 19. Scheme of reproduction "bystander effect" in the transmission of signals through the culture medium.

We evaluated the ability of the culture medium of exposed animal cells to induce an increased number of single-strand DNA breaks (SSB DNA) in the same cells obtained from unexposed animals. Level of SSB DNA in vitro in different kind of obtained cells was determined using the method described of labeling of DNA by fluorescent dye picogreen with subsequent evaluation of the rate of its splitting [Elmendorff-Dreikorn K., Chauvin C. & Slor H. 1999]. Results were presented as the coefficient of unwinding of the DNA helix (SSF), which was calculated at 20-minute exposure the DNA double helix (dsDNA) with untwine the buffer as follows:

SSF = log (% dsDNA in sample /% ds DNA in the control).

Confirmation of participation molecules free-radical nature in the transfer of "bystander" signals

Melanin-glucan complex (MGC) received from higher basidiomycetes Fomes fomentarius, was used as an antioxidant to confirm the involvement of molecules of free-radical nature to the transmission of signals from IrC to NirC after external influence of y-rays (the model of the single and chronic exposure of mice).

Melanins are amorphous pigment of dark brown and black, they are widespread in nature and are found in virtually all groups of organisms. Melanins contain carboxyl, carbonyl, hydroxyl, amine and phenol functional groups. Because of this molecule melanin can simultaneously interact with both anions and cations with, ie to be donors or acceptors of free (See Figure 20) electrons and thus carry out electron transport functions. Melanin is also able to absorb photon’s energy [Riley P. A. 1997]. These substances are characterized by the presence within their structure of unpaired electrons and possess the properties of stable free radicals. Melanin is not only absorb a variety of radiation, but also neutralizes and eliminates harmful for the cells of free radicals formed under action of ionizing radiation and some chemical substances on living organisms.

Study protector ability of microorganisms the presence of melanin in them almost all the basic mechanisms of reparative DNA repair revealed. Experimental results showing the increase in DNA polymerase and DNA ligase activity melanin mushrooms under UV irradiation were obtained by [Sidorik E. P., Druzhina M. O. & Burlaka A. P. 1994]. Melanin pigment has a high gene protective activity in acute exposure to ionizing radiation in a wide range of doses. It was established that melanin effectively reduces the frequency of mutations induced by ionizing radiation in both somatic and germ cells [Mosse I. B. 2002]. Ability of melanin to reduce almost to control level frequencies to genetic lesions, which are transmitted from generation to generation and accumulation in populations in the form of "genetic load" is unique [Mosse I. B., Lyach I. P. 1994]. For the first time is shown the principle possibility of effective protection of animal populations irradiated over many generations by means of melanin (in Drosophila studied 150 generations, in mice — 5) [Mosse I. B., Dubovic B. V. & Plotnikova S. I. 1996]. Radioprotective efficiency of melanin was higher in the chronic exposure than for acute irradiation conditions [Mosse I. B., Kostrova L. N., & Dubovic B. V., 1999]. Daily oral administration of melanin in a dose of 10 mg/kg to pregnant females rats eliminated the functional deficiency of physical and emotional development, detectable in the progeny at the antenatal y-irradiation at a dose of 1.00-1.25 Gy for the entire period of pregnancy. Conclusion of the radioprotective effect of melanin on cytogenetic and embryotoxic effects of low doses of ionizing radiation have been done on the basis of the data [Mosse I. B., Zhavoronkov L. P. & Molofey V. P. 2005]. Melanins are capable of forming complexes with metals, including radioactive elements. The ability of synthetic melanin to accumulate radioactive elements such as 111In, 225Ac and 213Bi had shown [Howell C. R., Schweitzer A. D. & Casadevall A. 2008]. The possibility of the creation of radioprotective agents on the basis of melanin in the result of research was mentioned [Dadachova E, Ruth

A. & Bryan R. A. 2008, Schweitzer A. D., Robertha C. Howell R. C. 2008]. For our experiments used the melanin-glucan complex whith a large number of paramagnetic centers (17 • 1017 spin/ g). This substance shows armipotent antioxidant properties in interaction with various types of free radicals [Seniuk O., Gorovoj L. & Zhidkov A. 2005].

Fig. 20. The structural formula of one of fungal melanin by [Riley P. A. 1997].

Results of the study remote signaling between cells in the external effects of prolonged y — radiation

The first attempts to search for distant signals generated by irradiation in vivo cells were taken from Balb/c mice that have known greater sensitivity to the action of IR. Quantitative analysis of DNA damage in a mammalian cell immediately after exposure to IR with low LET in a dose of 1 Gy has shown that it generates approximately forty DSB and MPS (DNA — DNA) 150 DNA-protein crosslinks about 2000 modification bases around 3000 AP-sites of damaged deoxyribose residues, SSB and alkali-labile sites [Von Sonntag C. 2006, Ward J. F. 1988]. It is believed that the sharp increase in single-strand DNA breaks (SSB) is directly correlated with the number of DSB. The ratio of SSB to DSB under the action of IR can correspond to the values 10 — 50 depending on exposure conditions and cell types of radiation [Oxidative Stress 1991]. Indicator of SSB DNA was used to assess the effects of ionizing radiation. In turn, the level of SSB DNA was determined by the value of the coefficient a DNA double helix unwinding in an alkaline environment.

Fig. 22. Influence of living medium from cells irradiated mice and from cells irradiated mice that received intraperitoneal injection of MGC before exposure on the levels of SSB DNA at cells non-irradiated mice (1st day after exposure at dose 5 Sv

As we can be seen from Figure 21. data in all types of cells used in the first day after irradiation dose of 5 Sv significantly increases the level of SSB of DNA. At the same time it is shown that the living medium obtained after three hours stay of IrCs on the first day after exposure is able to induce an additional level of SSB DNA in different types of cells from not-irradiated mice Balb/c. In certain cases, the change of this indicator reaches significant differences, particularly
in lymphocytes, hepatocytes and hematopoietic cells in bone marrow. While in splenocytes and astroglial cells revealed a clear trend towards increased levels of SSB DNA.

As shown in Figure 22 intraperitoneal injection of MGC, which has powerful antioxidant properties, into mice before irradiation procedure reduces the of BSE in all types of test cells and can serve as an argument in favor of the hypothesis about the important role of free radical molecules in the realization of this phenomenon. At the same time, it was shown that the transfer of "bystander" signals inside the irradiated Balb/c mice gradually decreases in all kinds of investigated cells during the first month after exposure and practically not detected 3,5 months after exposure (Figure 22). Receiving MGC before irradiation procedure is associated with a lower intensity "bystander" signaling in the period after exposure. The results of a comparative study of induction "bystander" signals in mice with different genetically determined radiation sensitivity under the same conditions of irradiation are shown in Figure 23.

□ Exposure □ Exposure after MGC application Fig. 23. Dynamics of development "bystander" effect for 112 days after MGC application before irradiation at a dose of 5.0 Sv

At least in mice with lower level of LD50/30 (Balb/c) induction of SSB in cellular DNA after exposure in living environment of the irradiated lymphocytes in various periods after of irradiation ranged from 150 to 200% when lymphocytes of mice more resistant to the effects of IR (C57Bl/6 with higher level of LD50/30) index of induction SSB in DNA of lymphocytes, respectively, was 2.5 and 5 times lower (Figure 24).

A correlation between higher index of LD50/30 and low induction as SSB DNA after irradiation, and induction of SSB DNA in modeling "bystander" effect in mice of C57Bl/6, can be explained by the presence in the cells of these animals melanized structures providing the black skin and fur. Antioxidants, photo — and radioprotective properties are a direct consequence of the free-radical structure of melanins, providing the opportunity to participate in electronic exchange of redox and radical processes. An attempt to identify "bystander" effect in vivo in mice of these lines under conditions of prolonged (over 231

days) of exposure on concrete bars with radioactive soil, have accumulated a total dose of external irradiation on the level of 0.29 Sv also been made. The data are shown in Figure 25.

Fig. 24. Different influence of the living medium of IrC on NirC obtained from mice with differ in genetically determined level of sensitivity to IR.

Fig. 25. Comparison levels of induction "bystander" signals in mice with different genetically determined radiation sensitivity under exposure during 231 day at dose 0.29 Sv.

Analysis of the results of this study convinces us that prolonged exposure of linear mice under the effect of у-fields of low intensity is associated with the induction of additional level of SSB in the DNA of cells of different origin. In this case, the phenomenon of amplification of radiological effects by means of "bystander" signals occurs. In this case, as with single-dose irradiation of 5.0 Sv "bystander" effect more clearly defined in biological mediums of mice with high sensitivity to the effects of ionizing radiation — line Balb/c. So, placing the NirC into living environment of the IrC Balb/c mice induces an increase in the level of SSB DNA in
different cell types from 33 to 44%, while a similar replacement of living medium in cells of mice C57Bl/6 changes the indicated index in the lower range — from 27 to 32%.

Detecting the ability of IrC to change the state of neighboring cells, through which not been laid tracks of ionizing particles, can in some extent explain the peculiarities of clinical realization of radiation effects at low doses of ionizing radiation. Identification BSE in liver tissue is an additional argument in favor of accepting the reality of the existence of diseases of hepato-biliary system of radiation origin. It was showed the development of the processes of autoimmune dysfunction in liver tissue during three-month exposure with accumulation total dose at 10.0 cSv in model studies on linear mice Balb/c [Kovalev V., Krul N., Zhezhera V. 2010]. It is known that one of the responses the cell to irradiation is the destruction of the cells with the loss of a specific morphology and functional activity.

Nowadays are known several forms of death in cells that depend on the production of ATP — apoptosis, necrosis and reproductive death. Postradiation apoptosis is characterized by maintaining the integrity of cell plasma membrane and the lack of contact, the intracellular content with cells of the immune system. In the end, remnants of apoptotic cells in tissues are removed by brushing up exfoliation in intraorganic space and subsequently excreted from the body. Mitochondria are the most sensitive to radiation cell organelles for several reasons: the practical absence of reparation and histone proteins that protect DNA, as well as the minimal activity of the enzymes to "cut out" and replace oxidized DNA regions [Anderson S, Bankier A. T. & Barrell B. G. 1981, Berehovskaya N. N., Savich A. V. 1994]. In the cell inhibits the synthesis of ATP because degradation and loss of mitochondria. But at lack of ATP, in particular energy-dependent mechanisms of apoptosis may be switched off in connection with exposure to IR. In this case cell necrotizing with the loss of integrity of the cell membrane and release of macromolecular components (alanine aminotransferase (ALT), aspartate aminotransferase, etc.) in the intercellular space. Necrosis caused an immune response in the form of inflammation — leukocyte infiltration of damaged tissue, the accumulation of interstitial fluid with subsequent induction of specific immune response to unmasked and recognized by lymphocytes intracellular components. Thus, the cells from renewable tissues which are sensitive to IR (epithelium of the gastrointestinal and urogenital tract, respiratory tract), regularly die as a result of intense radiation exposure. Their contents are subsequently released in extracellular space and blood. Taking into account the above mentioned thoughts we additionally determined the levels of intracellular enzymes ALT levels in peripheral blood of different animal groups — the control and chronically exposed mice, and mice immunized with liver-specific lipoprotein (LSP). The preparation of liver-specific lipoprotein (LSP) has been isolated from the liver of syngeneic mice by the method described by McFarlane I. G. [McFarlane I. G., Wojicicka B. M., & Zucker G. M., 1977]. It is a mixture of antigenic determinants of the substrate from the membranes of hepatocytes. Because of their lability, some proteins that are part of the LSP, in particular the asialoglycoprotein receptor [Treichel U., Schreiter T. & Zumbuschenfelde K. H.M, 1995] under certain conditions, including under the influence of small doses of radiation may acquire properties of autoantigens.

The one group of mice was immunized LSP to confirm the immunogenicity of the resulting substance. The preparation of LSP has been isolated from the liver of syngeneic mice by the method described by McFarlane I. G. [McFarlane I. G., Wojicicka B. M. & Zucker G. M. 1977]. The final concentration of protein in a preparation isolated by the method of Bradford [Bradford M. M. 1977] was 2.8 mg/ ml. The immunization scheme described in [Ryabenko D. V., Sidorik L. L., Sergienko O. V. 2001] was used. Increased serum level of this enzyme is considered a sign of inflammation in the liver — hepatitis, because a large amount of ALT released from the destructive cells of the body. Data obtained in this experiments are show in Figure 26.

Fig. 26. Dynamics of serum levels of alanin aminotransferase in control, irradiated and immunized of LSP of mice Balb/c.

Fig. 27. Dynamics of serum levels of AuAB to LSP in control, irradiated and immunized of LSP of mice Balb/c.

As shown by data presented in this Figures 26 and 27 a gradual increase in the concentration of ALT in the bloodstream is registered in all groups of animals observed. Increased serum levels of ALT in the mice of the control group reached significant difference in the fourth month of life and reflects a legitimate age-related changes of the liver associated with aging.

A clear tendency to increase this index in the group of animals treated with an immunogenic complex of proteins from the membranes of hepatocytes (LSP), when compared with that of control animals may be explained by stimulation of the processes of destruction of hepatocytes, mediated by immune mechanisms. More significant increase in the rate of ALT in blood is detected in irradiated mice (a significant increase in ALT is determined at the end of the first month of observation), and especially in mice immunized against membrane liver antigens contained in the LSP. Serum ALT levels increase in this group of experimental mice at the end of the second week after immunization began. Noteworthy the fact that the 100-day low-intensity radiation fields of fuel "hot particles" is quite effective in serum levels of ALT. Mice immunized with only one week ahead of irradiated mice to achieve a statistically significant increase of levels of this index compared with baseline. As follows from these data that a certain baseline level of AuAB to LSP is detected in the sera of control animals, and shows a tendency to increase during aging in mice. Amount of AuAB to LSP in immunized animals progressively increased and by the end of the first month of immunization reaches a peak and then decreases slightly and stabilizes over the next two months. In irradiated animals also determined by positive changes of serum levels of AuAB to LSP in the process of dose accumulation. In this group levels AuAB LSP to gradually increase and a tendency to increase AuAB remains at least during the observation period.

Registered positive changes of serum levels of ALT and of AuAB to LSP correlated with pathomorphological changes in liver tissue. In Figure 28. shows the morphological pattern of liver healthy 2-month old animal from the control group. Globular structure of the organ with well-differentiated trabeculae, which extend radially from the portal vein, clearly visible in the picture. The boundaries of cells and nuclei are well differ over the whole area of slice. In Figure 29 shown a slice of liver tissue with beginning an inflammatory process that is still impossible to differentiate as an autoimmune process. In figures 30 and 31 shown

Fig. 28. Morphological picture of liver healthy 2-month old animal from the control group.

deeper degenerative processes in the parenchyma with formation of lymphoid infiltrates. In a more favorable course of the process next sections of lesions appear regeneration areas. At high intensity the process changes in the tissues may be irreversible.

Fig. 29. Dystrophia of hepatocytes in violation of the architectonics of organ and the destruction of the nuclei. 1 — a violation of the architectonics of the lobules, blurred boundaries (irradiated mouse).

Fig. 30. Around blood vessels (or periportal) small clusters of mostly round the nuclear cell (lymphoid) elements. Stasis of blood within the vessel. More intensively painted the nucleus of cells (irradiated mouse).

Thus it was shown that exposure to IR can modify the immune status of the organism with the breakdown of immune tolerance and the subsequent emergence of autoimmune hepatitis. Its characteristic feature is the launch of autoimmune reactions against their own membrane antigens not only in the liver but also in other organs. The presence of long-term in excess of the background radiation pressure associated with the abolition of immune tolerance and the creation of conditions for the development of autoimmune reactions, in particular against antigens of liver tissue. As well known in turn the activity of autoimmunity is a favorable condition for the transfer of persistent infections in the active state and stimulation of vegetation as a saprophyte and pathogenic microorganisms was increased.

Fig. 31. Granulomas in the periportal areas of the parenchyma. 1 — Formation of granuloma on site infiltrate, 2 — Regeneration and repair of hepatocytes. (Irradiated mouse).

The impact of single external irradiation at a dose of 5.0 Sv for the transfer of intercellular signals in the bone marrow of mice Balb/c.

Nowadays it is known that hematopoietic stem cells (HSC) have higher radiation sensitivity than other cellular self-renewal systems. This conclusion was made by scientists on the basis of experimental works that showed the presence of the damage to hematopoietic progenitor cells not only due to the effects of the high doses but also upon action of the low doses of IR [Muksinova K. N., Mushkacheva G. S. 1995, Serkiz Ya. I. 1992].

It was shown that regeneration of the maturing cell pool and renewal of their quantity in the peripheral blood is determined by the completeness of the progenitor cell clone recovery. During the bone marrow regeneration period it was determined that both the proliferation of HSC is increased and the transition time of the maturing cell elements shortens [Bilko N. M. 1998, Bilko N. M., Klimenko S. V., Velichko E. A. 1999, Tavassoli M. V. 2008]. After acute period of the damage to the hematopoietic system regeneration phase follows, which is dependent on the viability of the stem cells, their migration ability in to the most affected areas of the hematopoietic tissues, time of proliferation and maturation of the committed progenitors and quantity of the functional mature cells [Shouse S. S., Warren A. S.L., Whipple G. H. 2004]. Critical for recovery of the hematopoiesis are quantity and quality of the HSC that recovered after irradiation. Possibility of hematopoiesis recovery is observed if more than 5% of the stem and progenitor cells remain intact and carry on proliferation and differentiation [Bond V. P., Fliedner T. M., Archambeau J. O. 2007]. If their level falls below this critical value, hematopoietic system can be exhausted due to lack of the stem cells capable of regeneration [Down J., Van Os. R., Ploemacher R. 1991]. Main proliferation stimulating factor of the HSC, which remain in the dormant state, is reduction of their quantity [Serkiz Ya. I., Pinchuk L. B. 1992]. Decisive role in the regulation of the recovery of the polipotent hematopoietic progenitors belongs to the microenvironment that upon interaction with HSC supports stability of their quantitative parameters in the physiological conditions and supports its recovery in case of injures [Hall E. J. 1991, Hall Mauch P., Constine L., Greenberger J. 2005]. Increase in the proliferation activity of the HSC is observed starting after irradiation exposure at the doses of 0.2 — 0.3 Gy [Grande T., Varas F., Bueren J. A. 2000].

The mechanism of the microenvironment influence on the hematopoietic system is still not fully determined. However, today it is known that its elements control the processes of hematopoiesis via production of the cytokines as well as by direct cell-to-cell contacts between HSC and microenvironment. Membrane-associated contacts serve for the transfer of the required molecules, homing and migration of the progenitor cells to the specific sites of the hematopoietic tissue and transport of the hematopoietic growth factors [Cronkite E. P., Inoue T., Hirabayashi Y. 2003]. Cultural investigations of the bone marrow (BM) indicated that despite the normalization of the quantitative parameters changes of the ability of hematopoietic elements to colony-forming had reduced character during prolonged periods of time with prevailing eosinophilic and neutrophilic colonies [Bilko N. M. 1998].

For the determination of the distant intercellular transfer of the post-radiation signals between the cells of irradiated animals a novel method of in vivo culture using diffusion capsules (DC) was described [Bilko N. M., Votyakova I. A., Vasylovska S. V., 2005]. Investigations were done of the Balb/c mice (Figure 32). The 16-hour model of exposure was used (see Protocol modes of mice irradiation). Animals were divided in to three groups: 1st group was irradiated without the use of radioprotector, 2nd group of animals received melanin — glucan complex prior to irradiation and the 3rd group was non-irradiated control.

Fig. 32. Groups of experimental animals

Further each group was separated into the subgroups of the donors (3 animals) and recipients (3 animals for each donor and 2 capsules per recipient). Donor animals were sacrificed on the day 1, day 7, and day 30 after exposure and bone marrow cells were extracted from the femur. In each case, colony-forming activity (CFU) in the culture was determined by injection of the 1x10s cells into the inner cavity of the diffusion capsule in the semisolid (0.33%) agar Difco. Diffusion chambers (DC) permit free diffusion of the peptide factors; however, they allow avoiding any contact of the cultured material with the immune system of the recipient. Each animal was implanted with two DC into the peritoneal cavity under Sagatal narcotization. Animals were retained in the conventional vivarium conditions with 12-hour light/dark cycle illumination and free access to food and water. After 12 days implantation DC were extracted from the recipients and investigated under the inverted microscope for the CFU activity as indicated by formation of the colonies or clusters of the proliferated HSC.

The groups of cells less than 20 cells were considered as clusters, while groups of cells from 20-40 were considered as large clusters, and all cellular aggregates above 40 cells were counted as colonies (see Figure 33 and 34).

Fig. 33. Granulocyte-macrophage colony of mouse bone marrow culture of Balb/c, irradiated with a dose of 5.0 Sv. Inverted microscope, increase 200.

Fig. 34. Granulocyte cluster of mouse bone marrow culture of Balb/c, irradiated with a dose of 5.0 Sv. Inverted microscope, increase 400.

Cultured material was extracted from the inner cavity of the DC and individual colonies were picked up for the preparation of the cytospin slides and Pappenheim staining for identification of the cell types. Obtained data indicated that BM of the animals of the 1st group was affected by the IR. Cell aggregate numbers were on average 24 colonies and 48 clusters in the cultures of the 1st day after exposure. Almost no colony-forming activity was observed in the culture of 7th and 30th day after exposure, that was indicative of significant suppression of the bone marrow function by the IR. At the same time in the culture of bone marrow cells that were obtained from the animals, which received MGC prior to exposure, the average colony count was 32 with 86 clusters at the 1st day after exposure, 45 colonies and 112 clusters on the 7th day after exposure, and 80 colonies and 136 clusters on the 30th day after exposure.

These results may indicate that MGC is able to protect the population of the bone marrow stem cell population for the influence on the IR and stimulate recovery after irradiation at dose comparable to the LD50/30. This conclusion can also be supported by increase of the quantity of the CFU in comparison to the 1st group of animals that have not received MGC. Implantation of the normal BM into the organism of the irradiated recipients at the 1st day after exposure after culturing resulted in formation of 114 colonies and 386 clusters on average. In the cultures of the 7th and 30th days such proliferation activity was observed, that it was not possible to determine individual colonies or clusters. There is indicative of a significant stimulation of the release of the compensatory signal substances by the radioresistant stromal cells of the BM, which highly stimulated the recovery of the radiation — damaged BM of the recipient. Normal bone marrow cells implanted into the irradiated mice treated with MGC yielded in 84 colonies and 192 clusters in the cultures of the 1st day after exposure, 52 colonies and 548 clusters on the 7th day, and 106 colonies and 302 clusters in the 30th day post exposure cultures (Figure 35).

Fig. 35. Determination of the CFU activity of the bone marrow derived stem cells: Group 1A — irradiated bone marrow culture in normal recipients; Group 2A — irradiated BM protected with MGC in normal recipients; Group 1C — normal bone marrow culture in irradiated recipients; Group 2C — normal bone marrow culture in irradiated recipients treated with MGC.

Such decrease in the CFU activity is indicative of less apparent stimulation of the compensatory factors release as a result of the radiation exposure due to protective effects of the MGC on the bone marrow cells so that less factors are required for reparation and therefore less factors are available for stimulation of the CFU activity in the DC.

Results of the in vivo culture of the bone marrow cells indicated that colony-forming activity of the hematopoietic progenitor cells of the animals non-treated with MGC prior to radiation exposure was significantly lower if compared to the control while treatment of the animals with MGC had increased the functional activity of the bone marrow cells.

Therefore upon irradiation exposure quantity of the hematopoietic stem cells is decreased and as a result, stromal component of the BM starts to secrete large quantities of cytokines and growth factors, which are able to stimulate HSC to active proliferation [Goldberg E. D., Dygai A. M. 2001]. Clearly, in animals irradiated at doses of 5.0 Sv, production of growth factors was blocked by excessive amounts of free radicals which are formed when passing through biological tissue of ionizing particles [Timofeev-Resovskii N. V. 1963]. At the same time, free radicals can quickly neutralized by the MGC in mice receiving radioprotector. Improved products of growth factors caused the hyperproduction of bone marrow stem cells. On the 7th day after exposure in the bone marrow culture of the irradiated animals, a deep depression in the CFU activity was observed. Finally pretreatment of animals with MGC resulted in the significant increase of the CFU activity. These data indicates that in the BM of irradiated animals the pool of later mononuclear cells was rapidly exhausted that resulted in the decrease of the CFU activity [Bilko N. M., Klimenko S. V., Velichko E. A. 1999]. In the group of animals pretreated with MGC prior to exposure, CFU activity in the BM increased during the experiment. On the 30th day post exposure, CFU activity of the bone marrow stem cells remained low and in the animals that have not received MGC, and in the animals treated with this radioprotector these values were significantly higher.

Obtained data may be indicative of the gradual recovery of the BM by implementation of the so called "golden reserve" of the stem cells that during radiation exposure were positioned in the crypts of the stroma and therefore remained undamaged [Tsyb A. F., Budagov R. S., Zamulaeva A. I. 2005]. Such significant effects of the MGC on the numbers of colonies and clusters in the DC cultures indicates decrease quantity of the stimulating bystander signals and strong radioprotective properties of the MGC, however the exact mechanism of action remains to be fully elucidated.

Concluding on the obtained experimental data it is clear that MGC is a strong radiation protector that helps to avoid consequences of the LD50/30 dose of irradiation at the level of HSC by affecting quantity of available growth stimulating factors that are commonly associated with radiation-induced damage to the BM.