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The method of DNA foci is based on an important property of some proteins toá ôrecognizeö the appearing DNA DSBs in nuclei and participate in the progression of the repair process. One of the initial stages of the formation of cell response toátheáappearance of a DNA DSB and activation of the repair systems isátheáphosphorylation of the H2AX histone. A phosphorylated histone, which is called H2AX, can be detected near a DNA DSB. It is a signal attracting other proteins toá theá DNAá DSB appearance sites. H2AX histone phosphorylation events can be visualized asáisolated nuclear foci by the immunostaining method. The immunostaining principle isábased on a specific binding of antibodies to antigens. For each protein, oráantigen, an antibody (a primary antibody) can be synthesized that would be specific only to this protein. The primary antibody binds with the studied protein; then, aá secondary antibody specific to the primary one binds with this primary antibody. Theá secondary antibody carries a fluorescent label, which allows the studied protein toá beá visualized. Besides the H2AX histone, this method allows visualization ofásome proteins participating in DNA DSB repair (like 53BP1).
Using the immunocytochemical staining technique and confocal microscopy, an international team ofá radiobiologists (A. V. Boreyko, L. Jezkova, S. Kozubek, M. Falk, M. G. Zadnepryanets, E. A. Kruglyakova) obtained three-dimensional images of human fibroblast nuclei irradiated with 60Co gamma rays (LETá0.3ákeV/m) and accelerated 11B ions (LETá135ákeV/m). For studying the kinetics of 11B ion-induced DNA DSB damage repair, the samples were irradiated normally to the cell monolayer. Irradiation atá aá small angle (10) allowed an analysis of the formation and structure of clustered DNA damage along the ion track. For the quantitative evaluation of DNA damage induction and repair, the co-localized H2AX and 53BP1 foci, which are the DNA DSB markers, were calculated.
In these experiments, the kinetics was studied of the formation and elimination of the H2AX/53BP1 foci induced in fibroblast nuclei by 60Co gamma rays and accelerated 11B ions. It was shown that in human fibroblasts accelerated 11B ions induce more H2AX/53BP1 foci than 60Co gamma rays. For gamma irradiation, the maximal radiation-induced H2AX/53BP1 foci yield is reached oneáhour after exposure (~ 25áfoci/cell); after four hours, most of the foci (~ 80%) are eliminated.
Foráaccelerated 11B ions, the maximal yield of these foci is observed after 45áminutes ofápostirradiation incubation (~ 72 foci/cell). 24áhours after irradiation, theáamount of theáradiation-induced foci was much higher in the cells exposed to accelerated 11Báions than in the cells exposed to 60Co gamma rays, which indicates that the accelerated ion-induced damage is more complicated. The different character ofáDNA DSB induction by gamma rays and accelerated heavy charged particles was illustrated by comparing the results of the 60Co gamma ray and 11B ion irradiation atá1áGy, theábeam hitting the sample surface perpendicularly and at 10 in different exposures. For the angle of 10, it was found that a particle, when passing through aánucleus, produces a track consisting of several neighboring foci; and clustered DNA lesions develop along the track in the first minutes after irradiation.
The different character of DNA DSBs induced by electromagnetic radiations and accelerated heavy charged particles and decreased cell repair ability after exposure toá heavy ions determine the character of the apoptotic cell death display (E. V. Baranova, A. V. Boreyko, I. I. Ravnachka, M. G. Savelyeva, S. I. Stukova). Asá is known, DNAáDSBs are the initiating signal of apoptosisáŚ programmed cell death.
The quantitative and qualitative differences in DNA DSB induction by ionizing radiations with different physical characteristics must show up in cellsĺ apoptotic response. Indeed, it is clearly observed in experiments on human blood lymphocyte irradiation with gamma rays and accelerated oxygen and neon ions (LETá170 and 180ákeV/m, respectively).
The Radiation Cytogenetics Group carried out the research in several fields.
The biological effectiveness of JINRĺs therapeutic proton beam was estimated;
investigations were started of the individual radiosensitivity of the human cell chromosome apparatus, mutagenic effect of ionizing radiations on mammalian cells, and genome instability.
As is known, proton therapy is one of the most promising areas of modern radiation medicine. A therapeutic proton beam was constructed at the Phasotron ofá theá Laboratory of Nuclear Problems, JINR; it has long been used for radiation therapy. Its effectiveness on human cells was estimated by cytogenetic methods. Asá aá model, human peripheral blood lymphocytes were used. Whole blood samples (cells in theáG0-phase) and a culture of lymphocytes stimulated to divide were irradiated at the times of different phases of the cell cycle. The cells were irradiated at theá170-MeV proton beam adjusted to perform proton therapy at two points of the depth dose distribution: at the place of the beam entering the object (LET ~ 0.5á keV/m) and near the modified Bragg peak (E ~ 0ľ30á MeV; the LET spectrum is up toá 100á keV/m). Specific quantitative and qualitative features ofátheáresponse of human peripheral blood lymphocytes to irradiation were revealed based on cytogenetic indicators. It was found that, by the criterion of the aberrant cell formation frequency and total chromosome aberration yield, the relative biological effectiveness of Bragg peak protons is ~ 1.2 for irradiation in the G0-phase of
the cell cycle. It was determined that G2-phase lymphocytes have the highest radiosensitivity to Bragg peak protons; this conclusion was based on different indicators:
the longest (upátoátenáhours) division arrest, a high frequency of the formation of cells with chromosome aberrations and the highest total chromosome yield, a sharp increase in chromosome fragmentosis (up to 85% of the total number of aberrations), and aá high frequency of the formation of cells with multiple chromosome aberrations. Itá was established that the pronounced changes in the ratio of different types of chromosome aberrations take place when G0- and G2-lymphocytes are irradiated with Bragg peak protons: a high level of chromosome-type aberrations with the prevalence of exchange aberrations is replaced by a high level of chromatidtype aberrations with the prevalence of fragments. Effectiveness coefficients of Bragg peak protons were obtained. Takingáinto account the most radiosensitive fraction ofálymphocytes (G2-lymphocytes) asáopposed to nondividing ones, the effectiveness coefficients ofá170-MeV protons areá~ 1.45 on the average.
A cycle of studies was conducted on the individual radiosensitivity of the human cell chromosome apparatus and biological dosimetry. A series of experiments were performed to study individual dispersions of the genetic structure damage distribution in chromosomes 2, 8, and 14 (human peripheral blood lymphocytes) depending on radiation LET. The accelerated ions used included 11B, 17Li, and 20Ne. Theáresults of the experiments show that the interdonor differences can underlie the biodosimetric error in determining the received radiation dose. Moreover, the ratio of centric ring and dicentric yield in chromosome 2 can be used as a reference quantity toáevaluate the radiation dose for high LET.
In this research, significant differences were revealed in the radiosensitivity ofádonor blood samples irradiated in the G0- and G2-phases of the cell cycle. Overall, the results show that for accelerated charged particles interdonor sample variability in the chromosome aberration frequency is higher than for gamma rays. Differences were observed between donor radiosensitivity levels evaluated by the usual metaphase method and premature chromatin condensation methodá(PCC). It was established that sensitivity to high-LET radiations is individual for each donor.
With the use of different cytogenetic analysis techniques, fingerprint estimations of the dicentric-to-ring and complex-to-simple aberration ratios (theáF andáC frequencies, respectively) revealed differences between donors with respect to these indicators. It was found that theáF factor is dose-dependent for gamma radiation, while no dependence of F on the dose and fixation time was observed for charged particles.á A combined analysis with theá PCC and fluorescence iná situ hybridizationá(FISH) techniques (PCC + FISH) showed that there is a clearáF andáC factor correlation with LET. It was established that the C factor is more variable between the donors thanáF. These results explain well the differences between the data obtained at different laboratories worldwide and confirm the efficiency of the PCC + FISH technique for evaluating the quality of radiations in biological dosimetry.
The Radiation Cytogenetics Group studies the biological effects of low doses of ionizing radiation (O. V. Komova, P. V. Kutsalo, E. A. Nasonova, N. L. Shmakova, T. A. Fadeeva). It was found earlier that above 30ácGy damage yield linearly depends on the dose, which is in full agreement with the generally accepted concept. Forálower doses, this dependence is nonlinear. In the beginning of the dose curve, abnormally high cell radiosensitivity was observed (damage yield per unit dose), which is followed by an increased radioresistance region, where chromosome aberration yield, in fact, depends inversely on the dose. The maximal effect is 2ľ3 times higher than the control; it is reached at ~ 5ľ7ácGy (the hypersensitivity peak). With further increasing the dose up to ~ 10ľ15áGy, the chromosome aberration frequency sharply decreasesáŚ in some cases, practically to the control level. Similar results were obtained for the irradiation of human blood lymphocytes with carbon ions, whereáaádistinct hypersensitivity peak was observed at the doses of ~ 5ácGy. It should be noted that in lymphocytes of some donors no hypersensitivity or increased resistance was observed in the beginning of the dose dependence of the chromosome aberration yield, which allows this phenomenon to be considered an individual feature of these donors.
It was also found in research on lymphocytes that independently on the radiation quality, chromatid aberrations are the main type of chromosome damage inátheáhypersensitivity region. According to the concepts of classical radiobiology, aberrations of the cromatid type are not induced by radiation in nonstimulated lymphocytes.
Atá the same time, they make the main contribution to spontaneous mutagenesis, which, as is known, is determined by the effect of endogenous reactive oxygen species (ROS). Mitochondria, in whose respiratory chain 2ľ3% of oxygen is converted into the superoxide anion in the process of normal metabolism, are the main ROS source in the cell. As a result of its interaction with a number of cell substrates, aáspectrum of secondary active radicals are formed, among which the most dangerous for the cell are the hydroxyl radical and hydrogen peroxide. It was shown earlier that ionizing radiation can cause a sharp increase in mitochondrial superoxide anion yield during several seconds after irradiation (the so-called mitochondrial oxidative stress). Evolutionally, a powerful protein complex formed in cells which protects them from endogenous ROS, but under irradiation its potential may not be sufficient to neutralize such a significant amount of the produced radicals; therefore, additional cytoprotective pathways have to be activated. All these facts made up theábasis of the hypothesis on the mechanisms of the action of low doses of ionizing radiation. Its main points are the following: a)áhypersensitivity observed inátheábeginning ofá theá dose curve is caused by an increase in the yield of oxidative damage in cellá DNA, which results from radiation-induced ROS amplification in cell mitochondria; b)áincreased radioresistance observed in cells with further increasing theádose isátheáconsequence of the activation of the cytoprotective mechanisms aimed at suppressing oxidative stress. In the capacity of such a mechanism, a cascade of reactions leading toátheáactivation of the signal-regulated ERK protein kinase was examined. The cascade is triggered in response to an increase in the mitochondrial ROS yield and the action of radiation; it causes the enhancement of cell proliferation.
To test this hypothesis, different modifiers influencing ROS, the mitochondrial respiratory chain, and ERK were used. A change in the shape of the dose dependence of aberrant cell yield in the presence of these modifiers allowed the evaluation ofátheácontribution of the above mentioned processes to the phenomena of hypersensititvity and increased radioresistance at low doses of ionizing radiation.
In research on human breast carcinoma cells, it was shown that the substances having an effect on ROS eliminate hypersensitivity. They include the DMSO interceptor of free radicals, cyclosporine A (CsA)áŚ a blocker of ROS generation byámitochondria, and the SB203580 inhibitor of the p38 MAP kinase, which blocks theáprolonged generation of ROS by the NADPH oxidase. At the same time, antimycináŚ anáelectron transport inhibitor in mitochondria, which is widely used asáa ROS generator in biological systemsáŚ led to a yet greater increase in chromosome aberration yield at low doses. To clarify the protective role of ERK, two inhibitors suppressing its activity were used: PD98059 and U125. As expected, ERKáinhibition prevents an increase in radioresistance. At the doses of 7ľ8áGy, which corresponds to the maximum radioresistance of nontreated cells, the percentage ofáaberrant cells increases 1.5ľ2-fold. All these facts indicate that the activation ofáthis protein is aánecessary factor of cell protection when the constitutive cytoprotective systems cannot manage an increased number of oxidative lesions observed ináthe hypersensitivity region.
Overall, experiments with different modifiers influencing the endogenous ROS yield showed that these compounds, which have a high mutagenic potential, make aá significant contribution to chromosome damage induction in breast carcinoma cells atálow doses. This fact allows one to suggest that radiation-induced oxidative stress and its consequence, cell homeostasis disorder in general, can notably affect the irradiated cell fate.
Another field of studying biological effects at low doses of ionizing radiation is the research on the regularities in adaptive response induced in human blood lymphocytes by different doses of gamma radiation (2ľ15ácGy). As is known, adaptive response isáone of the specific effects of low doses of radiation. It consists ináan increase in cell and organism resistance to the subsequent exposure to greater doses ofá radiation. Adaptive response depends on many factors, including the primary and main dose values, dose rate, cell cycle stage at the moment of irradiation, and theátime between the primary irradiation and the subsequent irradiation with a higher dose. All these factors impose tight constraints on the adaptive response manifestation. Besides, iná repeated experiments on the same biological objects in
the same conditions, preirradiation with a low dose often resulted in opposite effects:
from pronounced adaptive response to the enhancement of the effect caused by irradiation with a high dose. It is thus doubtful that this is a universal phenomenon.
The aim of the study was toáevaluate the reproducibility of adaptive response and determine whether anáoptimal priming dose exists for any individual, which, as had been suggested, can depend on the organismĺs individual radiosensitivity in different low-dose ranges. An adaptive response research was conducted on three donorsĺ G0-lymphocytes in a wide range of gamma radiation priming doses. Irradiation with the major dose of 1áGy was performed in the G2-phase of the cell cycle. As the criterion, aberrant lymphocyte yield registered by the metaphase method was used.
Altogether, three experiments were conducted with six-month intervals.
The research confirmed a high degree of variability in adaptive response manifestationsáŚ between different donors and in different tests of the same donor. Itáwas shown that the individual radiosensitivity factor has no influence onátheáadaptive ability associated with the irradiation of cells at low doses. Moreover, itá turned out to be impossible to find optimal doses for any specific individual, theápriming irradiation with which would have had a radioprotective effect in each experiment.
Obviously, there is some stochastic factor that affects the radioadaptive response manifestation. Its nature is difficult to establish, because its mechanism is unknown.
Thus, adaptive response, due to its extreme instability, cannot be considered asáaáuniversal phenomenon that could be used in clinical practice or taken into account when evaluating radiation risks.
The Radiation Cytogenetics Group performs large-scale research on the mutagenic action of ionizing radiations of different quality on mammalian cells and theáproblem of genome instability (P. Blaha, R. D. Govorun, I. V. Koshlan, N. A. Koshlan).
Chinese hamster cells irradiated with protons (LETá0.22ákeV/m) and accelerated 11B, 14N, 18O, and 20Ne ions (LETá 50ľ153á keV/m) were used to study regularities in HPRT mutation induction. It was found that the manifestation of mutations depends on the time of seeding irradiated cells into a selective nutrient medium withá6-thioguanine (mutation expression time) and radiation LET. Foráaáfour-day expression, the frequency of spontaneous and radiation-induced mutagenesis was about 1.2 10-5. For a longer expression, the mutagenesis level increased approximately threefold, reaching a maximum. The maximum location depended onáaccelerated ion LET. With increasing LET, the maximum shifts towards longer expression times. In particular, the maximal level of mutagenesis was observed 11ádays after irradiation with 18O ions (LET ~ 116ákeV/m) and 23ádays after irradiation withá20Ne ions (LET ~ 153ákeV/m). These terms correspond to 40ľ50ácell generations (one Chinese hamster cell division cycle takes 11ľ12á hours).
During the identification and selection of mutant subclones, mutants were observed that grew slower than the intact control cells. The slowdown of the growth ofámany mutant subclones in a selective nutrient medium with 6-thioguanine could be determined by the appearance of mutations leading to a decrease in HPRT enzyme activity or to the synthesis of a lower amount of the HPRT enzyme. In these cases, the viability of the mutant population could be provided only by the cells that have no time to utilize the purine analog during their cell cycle. Also observed were nonstandard types of the growth of mutant subclones isolated from Chinese hamster cells irradiated with accelerated 18O ions (LET ~ 153ákeV/m) atá0.5,á1, and 2áGy. In the same growth conditions, some mutants show unusual morphology compared with the control cell population: the laced, chain, and stellar character of growth.
Emergence of colonies was observed before the formation of the mutant subclone cell monolayer. These signs can indicate the initiation of the malignant transformation of cells.
The Group of Lower Eukaryote Radiation Genetics studies regularities in the induction of mutations of different molecular nature in cultures of the unicellular yeast Saccharomyces cerevisiae by different types of radiation (N. A. Koltovaya). Several genetic systems are used which allow testing specific types of genetic structure damage. Base pair substitution is tested with two genetic systems which are based on the substitution of a nucleotide in critical amino acid codons in theáCYC1 gene: Cysá22 cysteine (CAA TGC CAC) and glutamic acid Glu50 (ATC GAA TTG). These systems allow testing all the transition and transversion types; they are constructed in such a way that the reversions can emerge only at the expense of true reverse mutations. The disadvantages of the CYC1 system include respiratory impairment, which itself can have an effect on mutagenesis. Besides, the reverting frequency can be affected by neighboring nucleotide sequences. In this connection, work was started with another test system: TRP5. The mutations in this gene do not disturb respiration, which allows selecting the revertants against the active respiration background;
and the surrounding of the critical codon of the TRP5 gene differs from the nucleotide sequence of the critical codon of the CYC1 gene. So, the data obtained with the latter test system allow generalization of the regularities in the induction of base pair substitutions by radiation.
Survival curves were obtained for all the haploid (YMH1-7) and diploid (YMH51strains of the CYC1 test system under ultraviolet (UV) light. The survival curves of the haploid and diploid strains are sigmoid. For the YMH53 diploid strain, UVinduced mutagenesis curves were obtained; they have a linear quadratic character.
With the survival rate of about 1%, the ATľTA transversion frequency increases 18-fold, reaching 10-8. Survival and mutagenesis curves were also obtained for six strains of the TRP5 test system under UV light. The strains do not differ inátheir survival rate, and their survival curves have a typical shape. Attempts to induce mutations in haploid strains of the CYC1 genetic system with UV light failed, while mutations were efficiently induced in the TRP5 test system, the GCľAT and ATľGC transitions prevailing in the spectrum.
For the CYC1 test system strains, survival and mutagenesis curves were obtained under gamma irradiation. The haploid strain survival curves have an exponential shape; the diploid ones are sigmoid. A linear (for haploids) and power (for diploids) dose dependence of mutation induction is observed. Gamma radiation efficiently induces all types of base pair substitution; at the survival rate of ~ 1%, the maximal mutation frequency was 10-6. In haploid strains, GCľCG transversions and GCľAT transitions were induced most efficiently; in diploid strains, GCľAT transitions and GCľTA transversions. The induction of base pair substitution by gamma radiation isánow studied on a second TRP5 test system.
To study regularities in the induction of frameshift mutations, two genetic test systems are used in which the strains carrying the lys2-Bgl and hom3-10 frameshift mutations in the LYS2 and HOM3 genes, respectively, reverted due to the omission of one or two nucleotides in the 5A or 4C tracks in the lys2-Bgl mutant and 7T inátheáhom3-10 mutant.
The analysis of the mutagenic effect of UV radiation revealed the following regularities. The gene mutation dependences on UV fluence are nonlinear for all types ofámutations. Frameshift mutations are induced by UV radiation equally efficiently in both test systems: LYS2 and HOM3. However, the genetic systems forá testing base pair substitution mutations are different, the induction of the GCľAT transitions taking place with the same efficiency as the induction of frameshift mutations inátheáLYS2 and HOM3 test systems. These results show that in the used test systemsáŚ LYS2, HOM3, and TRP5áŚ the context of the nucleotide surrounding ofátheámutations is the most suitable for testing frameshift and base pair substitution mutations induced by UV radiation. For gamma radiation, it was established that theádose dependence of frameshift mutation induction is linear.
To test the induction of extended deletions sized approximately several thousand nucleotide pairs, a plasmid system is used which, based on genetic methods, allows detecting the omission of DNA fragments with several genes. Five genes were inserted into a shuttle vector that had regulatory elements of plasmid support inábacterial and yeast cells. A large size of the plasmid and its nucleosome structure allow extrapolation of the obtained data on chromosome-type DNA. The size and localization of the deletion are determined with the electrophoretic and restriction analysis of plasmid DNA.
Experiments showed that deletions are induced by UV light. An exponential dependence of the deletion mutant frequency on the irradiation dose is observed, and the fraction of more extended deletions increases with increasing the dose.
Gamma radiation also induces deletion mutants; the dependence of their frequency oná theá dose is nonlinear. At the irradiation dose of 100á Gy, the mutation frequency wasá10-5. Research is conducted on genotype influence on deletion induction regularities. It was shown that UV light and gamma radiation induce deletions inátheárad53 mutant, the dose dependence of mutation yield being power-like.
Theárad53 mutation in the checkpoint gene leads to a decrease in the deletion mutation frequency; thus, the participation is proved of the RAD53 gene in double-strand break (DSB) repair by DNA DSB end joining.
These results were obtained in the conditions when one of the inserted genesá(URA3) was used as a selective marker, and the omission of the other four genes was studied. Mutants with different omitted gene spectra were isolated.
plDNA was taken from 20 mutants; the size and precise localization of deletions are being determined.
During acquisition of results on a testing system with disordered respirationá(CYC1), the issue emerged of the influence of respiration disorder on the lethal and mutagenic effects of radiation. Besides, a high level (up to several percent) ofárespiratory failure mutations is typical of budding yeasts, which is caused by mitochondrial genome mutations. For this reason, the influence of mitochondrial genome disorders causing respiratory failure on nuclear genome mutability was analyzed. Itáwas shown that respiration disorder caused by mitochondrial genome damage (rho- and rho0 mutations) decreases the survival rate under gamma irradiation, butáhas noáeffect on the survival rate under UV irradiation. Respiration disorder did not influence frameshift mutation induction (LYS2 and HOM3) by UV light and gamma radiation. In respiration mutants, however, the induced deletion mutant frequency increased both under UV light and gamma rays. In the plasmid system used in this work, deletions emerge as the result of repair by DNA nonhomologous end joiningá(NHEJ). Toáclear up the mechanism of this effect, further research is needed as to whether respiration disorder influences the efficiency of DNA DSB repair due to the blocking ofáATP energy molecule synthesis, or the formation of NHEJrepaired lesions decreases.
The Photoradiobiology Sector conducts research on radiation lesions iná mammalian eye structures (the lens and retina). The lens is a very radiosensitive organ.
As early as the late 19th century, it was clear that exposure of the lens to X-rays leads to its opacityáŚ that is, cataract development. The International Commission onáRadiological Protection (ICRP) established that the threshold doses for cataract development areá 2 andá 5á Gy for a single and fractionated exposure, respectively.
Theáanalysis of epidemiological data collected in recent years, however, allows suggesting that these threshold values are overestimated at least by a factor ofá 5ľ10.
For this reason, research has been stimulated in developed European countries and theáU.S. onáradiation cataract development mechanisms and cataract epidemiology in population groups which should not be included in the risk group according toátheámodern concepts.
50 years of the manned exploration of space showed that space flights are associated with an increased risk of cataract development in spacecraft crew members.
This problem is especially pressing since the idea of long manned flights beyond theáEarthĺs magnetosphere is considered. The main factor is the organismĺs exposure to the cosmic rays, which consist of different radiations. In the space flight conditions, it is practically impossible to provide protection from their main componentáŚ high-energy heavy charged particles. Thus, research on the mechanism of cataract development under such radiation is an important applied aspect of the radiation cataract problem.
The Photoradiobiology Sector conducts research on the mechanisms of radiation cataract formation (K. O. Muranov, M. A. Ostrovsky). It was established that, like in the case of senile cataract, the epithelium structure changes under irradiationá Ś cavities and defective cells emerge there, the capsule becomes thin, and oxygen concentration in the tissue increases. Fiber cell morphogenesis gets disordered; nuclei, mitochondria, and other cell organelles, which should normally be eliminated, remain in the formed cells. An increase in oxygen concentration and the functioning of mitochondria lead to the enhanced production of reactive oxygen speciesá(ROS), oxidative damage of the protein, and its aggregation. Full coincidence was found between the lens regions with an increased ROS concentration, protein aggregates, and opacities proper. Radiation induces additional ôdestructionö of the nuclear apparatus of epithelial cellsá Ś that is, yet greater disorder in fiber cell morphogenesis. Radiation action is summed with the natural aging of the lens. Irradiation with increasing doses leads to a proportional decrease in the lag period of the formation ofádefective fiber cells in the lens cortex and cataract development. This study resulted in an essential conclusion that the idea of a threshold radiation dose is unacceptable as regards cataract induction. Exposure to ionizing radiation only brings closer the beginning of cataract formation.
Among the targets of high-energy heavy charged particles in the lens, the protein and DNA molecules can be determined as the main ones. It is known that toádamage a protein by ionizing radiation, relatively high doses are needed. But the special structure of the lensáŚ namely, the absence of protein exchangeáŚ can result in longterm damage accumulation and, later, protein molecule denaturation. Moreover, hidden internal damage of the molecule can weaken its resistance to other damaging factorsáŚ in particular, ultravioletá(UV) light. For this reason, a cycle of research was performed to study the effect of different types of radiation onáL crystallin stability.
The main method consisted in studying the kinetics of this proteinĺs aggregationáŚ that is, the kinetics of the protein solutionĺs opacity under factors denaturing the protein. It was shown thatáL crystallin exposure to UV light leads to one-hit protein denaturation; in this case, aggregation kinetics is described in terms of clusterľcluster interaction. It means that the molecule accumulates internal damage, which in no way affects its properties, but upon reaching some dose, single-step denaturation takes place. First, denatured molecules form primary clusters with aásize of about 20ánm; then, clusters stick together to form large light-scattering aggregates. The action of the following types of ionizing radiation was studied: gamma rays and H, D, He, 12C, 7Li, and 11B nuclei. The most active were lithium and boron nuclei that is, radiations with the highest linear energy transfer. However, theádoses for which aádecrease in protein molecule stability was observed were quite high: 16áGy for these nuclei. It is obvious that such doses have no physiological sense asáthe lethal dose for humans is 10áGy. The data allowed concluding that epithelial cell DNA is the main target for radiation in the lens.
At the next stage, the lens in vivo became the object of research because the lifelong dynamics of its condition allows tracking DNA radiation damage.
In the natural environment, cataract results from organism exposure to many cataractogenic factorsáŚ in particular, UV light, malnutrition, smoking,áetc.áTherefore, atáthe next stage, the integrated effect of the main cataractogenic factors on cataract formation was studiedáŚ radiation, UV light, and age. In cooperation withátheáInstitute of Eye Diseases of the Russian Academy of Medical Sciences andáEmanuel Institute of Biochemical Physics of the Russian Academy of Sciences, itá was shown that theádevelopment of cataracts of different genesis (senile, UV, diabetic,áetc.) seems toábe underlain by the same mechanism. The influence ofádifferent damaging factors on the lensáŚ in particular, radiationáŚ manifests itself asáacceleration ofátheánatural process of senile cataract development (K. O. Muranov, M. A. Ostrovsky).
Radiobiological experiments on the retina involve molecular biological, morphological, and electrophysiological research techniques. As a brain part placed ináthe eye (Ramn y Cajal, 1901), the retina can rightfully be considered a model and object for studying the effect of radiation on the central nervous system. Research on the mechanisms of the effect of different types of ionizing radiation on the retina is of principal importance for evaluating the risk of postirradiation complications in the radiation therapy of the eye and brain as well as the real danger of long space flights. The latter is associated with the damaging effect of heavy charged particles of the galactic origin on the retina beyond the Earthĺs magnetosphere, the clinically apparent manifestations of which can develop months and even years afterwards.
Along with studying the mechanism of radiation cataract development, theáSector conducts research on the radiation-induced effects in experimental animalsĺ retina (Yu. V. Vinogradova, V. A. Tronov). Experiments were conducted to study the link between DNA damage and repair on the one part and, on the other, degenerative changes in the mouse retina induced by ionizing radiation (gamma and proton radiation) and the genotoxic agent methylnitrosoureaá(MNU). Gamma radiation induces mainly DNA single-strand breaks, which are uniformly distributed over the genome.
Protons are more efficient in the induction of double-strand breaks, which localize near the particle track. Double-strand breaks are lethal lesions because of their high efficiency in apoptosis induction in dividing cells. The methylating agentáMNU causes breakless lesions in DNA: methylated bases along with apurine and apyrimidineá(AP) sites. In the mid-1990s, a research team in Japan discovered MNUĺs ability to induce photoreceptor apoptosis in the retina after single intraperitoneal introduction in animals at a dose of 60ámg/kg. MNU was used as a positive test of apoptosis in the retina. Thus, the three used agents are associated with the main DNA damage types and their repair pathways.
The obtained results confirm the thesis that the mature mouse retina is highly radioresistant. Full DNA repair is observed after exposure to gamma and proton radiation at a dose of 14á Gy. Increased expression in retinal proteins that are associated with cell death (apoptosis) is normalized 12á hours after irradiation. By thisátime, radiation-induced DNA break repair is finished. Most likely, it indicates that theseáproteins facilitate DNA repair and damaged cell renovation rather than induce apoptosis.
Increasing the exposure dose to 25á Gy caused notable morphological changes in the photoreceptor layer of the retina. The changes include the degradation ofátheáouter segments of the photoreceptors and a decrease in the density and thickness ofá their nuclear layer. Degradation progresses in time and is connected with photoreceptor death, which follows the apoptotic pathway. Apoptosis is indicated by the increased expression of proapoptotic proteins. Thus, the relatively high radioresistance ofátheáretina and the active mechanism of postirradiation repair, which removes radiation-induced DNA breaks, point to the existence of the genotoxic threshold that determines a nonlinear character of the effect dependence onátheáirradiation dose.
In the genotoxic effect of MNU, dose threshold existence was also established.
Research on its connection with the genotoxic effect of MNU revealed two specific features of the retina that had not been described before. First, it is a highá DNA damage level in the mouse retina. In the increasing order of the DNA damage level, mouse organs are ranged as follows: lymphocytes liver brain retina. This order is the same for the degree of the oxygenation of these tissues.
The second peculiarity of the retina found in the Sectorĺs research is its capability of active repair, which removes most of DNA damage induced by radiation andáMNU but has no effect on the earlier spontaneous DNA damage.
Thus, these results confirm that in the differentiated retina cells, there is a genotoxic threshold for the used radiations and methylate. They also show that, like forátheádividing cells, repair is one of the causes of postmitotic retinal cell tolerance toáDNA damage. Another cause of this tolerance is a decrease in the physical size of the radiosensitive target to that of the transcribed locus of the genome. There are grounds to assume that the decisive role in the transformation of the originally permissive DNA lesions (postirradiation breaks along with modified bases andáAPásites emerging after exposure to MNU) into cytotoxic ones belongs to the topoisomerase 2ámolecules localized in the transcribed sites.
In recent research performed by the Photoradiobiology Sector, electroretinographyá(ERG) has been used as an overall physiological indicator of the retina's functional integrity. Recording an electroretinogram induced by white light pulses ofádifferent intensity allows obtaining a full picture of the lifetime activity of the mouse retina. It was found that the ERG profile is more sensitive to a genotoxic effect than the morphological and cell indicators. With this approach, it was found that theáretina is capable of adaptive response and recovery with respect to the functional activity indicator. The Sectorĺs current research is focused on a possible contribution ofáMller glial cells to the retina recovery. These cells make up a small population ofáretinal cells which, in response to traumatic stress, retain the ability to increase their proliferation, migrate to the outer retina layers, differentiate into photoreceptors, and produce endogenous neuroprotectors for the retinal photoreceptors.
The Laboratoryĺs Mathematical Modeling Group performs mathematical modeling of radiation-induced effects in cells of different organisms. As was already said, the initial aim of these studies was the development of mathematical models of molecular mechanisms of the induced mutation process in relatively simple biological objects like bacterial cells. Based on experimental data, an original model was developed that described the induced mutation process by detailed mathematical modeling of the key protein interactions during the specific response ofáE.ácoli bacterial cells to ultraviolet irradiation (SOS response) (O. V. Belov, E. A. Krasavin, A. Yu. Parkhomenko). Using this model, it was shown that the magnitudes and time locations of the protein concentration maximums and minimums depend oná theá ultraviolet radiation energy fluence. It was found that the concentration dynamics ofá the UmuD2, UmuD'2, and UmuDD' dimers is described by a curve with a local maximum, which shifts towards longer times with increasing the radiation energy fluence. Typical for the UmuD2 dimer is a decrease in the protein concentration atá the early stages of SOS system functioning. It was shown that if theáenergy fluence is greater than 30áJ/m2 for the UmuD protein and greater thaná49áJ/m2 forátheáUmuD2C protein, two concentration peaks are observed; their location depends on the magnitude of the radiation energy fluence.
In this research, for the first time a relationship was established between theá molecular mechanisms of the bacterial system of SOS response, translesion synthesisá(TLS) efficiency, and gene mutation yield. It was shown that an increase iná theá concentration of DNA polymeraseá V results in more errors in the course ofáTLS. With the use of the lacI regulatory gene of E.ácoli as an example, the dependence ofátheálacI- mutation frequency on the radiation energy fluence was calculated; the modeling results agreed with experimental data.
The research was based on the modern concept of SOS regulation, which suggests considering cell SOS response in terms of system biology. The aim of this approach was to provide insight into the biology of SOS response at the system level by formalizing the mechanisms of the protein interactions in the cell. The developed model approaches have two main features that make them applicable for the future development of the SOS regulation theory: first, they contain a descriptive functionáŚ that is, a topologic presentation of the system components and their relations; second, they can predict the dynamic behavior of a biological system in time.
For a more precise identification of the molecular mechanisms of fixing premutational lesions as mutations, approaches were developed to the detailed description of induced SOS response in bacterial cells E.á coli with the impaired translesion synthesis function. A dynamic change in the concentrations of the key proteins ofátheáSOS system for the recA, umuD, and umuC mutants ofáE.ácoli was modeled.
Then, to take into account the stochastic nature of biochemical interactions, aá SOS response model was developed based on the Gillespie algorithm, which had become widely used in modeling complex biological systems. The advantage ofáthisáapproach is a more correct description of protein interaction kinetics at the level ofáaáspecific cell at low energy fluence values ( 1áJ/m2).
Further work was concerned with mathematical modeling of other repair systems that have an influence on the magnitude of the bacterial SOS systemĺs inducing signal. In particular, with the use of a stochastic approach, a model was developed that describes the key processes of the excision repair of damaged bases ináE.ácoli cells (O. V. Belov, M. A. Kapralov). The mechanism of the removal of 8-oxoguanine modifications was modeled involving formamidopyrimidine DNA glycosylase (theá Fpgá protein), which has several types of activity. The proposed model included the description of repair processes like modified base transformation intoáAP sites, - and -elimination, 5'-deoxyribose phosphate residue excision, and DNA polymerase Iáand DNA ligase activity. Such a model, which takes into account the stochastic nature of the biochemical reactions, allowed predicting the kinetics ofáthe key enzymes and intermediate DNA states during excision repair. The modeling results agree with inávitro experimental data that characterize the initial stages ofáthe repair process involving the Fpg protein. Predicted were the dynamic change in the Fpg protein concentration, DNA polymerase I, DNA ligase, and metastable states during repair. It was found that the rate of some stages of the systemĺs functioning depends on the initial concentration of 8-oxoguanine.
In the course of this research, a particular example was considered of applying the proposed model to the repair of 8-oxoguanine lesions involving bifunctional Fpg glycosylase. It was shown that the model can describe an excision repair ofáE.ácoli ináthe general view with the participation of other DNA glycosylases, including multifunctional ones, in which case some of the activities typical of Fpg are realized byáadditional enzymes.
An important aspect of this work is the introduction of the magnitude equal toáthe fraction of lesions that were not removed at the Fpg-dependent stages ofárepair.
Thus, it is possible to do a probabilistic study of the excision repair stages atáwhich aásystem failure can happen. In fact, the research shows that the introduced magnitude can be a reliability characteristic of this repair system, and a search for similar parameters of other repair systems is an important approach to the evaluation ofárepair process reliability in whole.
The results of these studies were published in the cycle of works ôMathematical Modeling of Radiation-Induced Mutagenesis in Bacterial Cells,ö which won theá2011 First Prize of the journal ôParticles and Nuclei, Letters.ö The specifics ofá these works included using modern data on the role of genes and proteins participating inátheáregulation of the repair processes and obtaining results that can be verified experimentally. On the basis of the developed model concepts, a number of important regularities were predicted concerning the character of the expression of some proteins during the functioning of the inducible repair systems. The proposed approaches can be widely used to describe the acquired experimental and theoretical knowledge of the induced mutation process. In particular, clearing up the radiationinduced mutagenesis mechanisms in complex organisms and the human is hardly possible without a detailed analysis of the mutation process in relatively simple biological objects like bacterial cells.
In parallel, work was being performed on mathematical modeling of the repair process under accelerated heavy ions. The inducing signal of the SOS system ofáE.ácoli was evaluated for different types of accelerated ions. Formation of the main premutational DNA lesionsáŚ base damage, single- and double-strand breaks, and clustered lesionsáŚ was described quantitatively. DNA was considered as a linear target randomly positioned relative to the charged particle track. The model takes into account the character of the radial distribution of energy in the particle tracks, which is very important for assessment of delta electronsĺ role in damagingá DNA. Calculations confirmed that the character of the base damage yield dependence oná linear energy transfer is similar to that obtained for single-strand breaks. Base damage yield turned out to beá 4á times greater than single-strand break yield throughout all the calculated linear energy transfer range, which is connected with an effective increase iná theá thickness of DNA linear target. The dependence ofá DNA doublestrand break yield and clustered damage on linear energy transferá (LET) are described byáa curve with aámaximum, after which a furtheráLET increase becomes inefficient. The calculation results, which describe the total yield of clustered lesions (independent ofátheir type), are compared with experimental data on SOS induction potencyá(SOSIP) evaluated with a SOS chromotest. The calculated and experimental results agree well. Along with these studies, preliminary model calculations were performed that describe the main repair processes leading to the generation ofátheáinducing signal of the bacterial SOS system. In particular, preliminary quantitative estimations were obtained of the polá A-dependent repair of single-strand breaks, double-strand break repair by homologous recombination, and excision repair ofámodified bases. Theáproposed models take into account the possibility of different damage types transforming into the other ones.
The final stage of research on modeling induced mutagenesis in bacterial cells was the development of models substantiating the presence of additional elements in the hierarchy of the repair systems regulating the fixation of premutationáDNA lesions as mutations. On the basis of the constructed models, important conclusions were made on the role of mismatched base repair in the realization of radiationinduced SOS response. Described in detail was the interaction between SOS repair and mismatched base repair, which leads to the significant leveling of the mutagenic effect ofáDNA polymerase V. In earlier studies concerned with SOS response modeling, a discrepancy was revealed between a high level of mistakes emerging duringáDNA polymeraseáV functioning and the relatively low efficiency of the fixation ofáthese mistakes as mutation. It was assumed that there is an additional molecular mechanism decreasing the number of bases the matching of which took place witháthe violation of the complementarity principle in the course of DNA resynthesis on single-strand sections. Experimental results obtained over past several years allowed concluding that this mechanism is a mismatched base repair, which identifies and removes a noncomplementary nucleotide and then fills the formed gap. It remained unclear, though, how these two repair systems interact and how their mutual regulation is realized. The lack of understanding how these repair processes interact was caused, first of all, by the absence of a system view of the functioning of these mechanisms.
It became possible to resolve these issues by working out a mathematical model of mismatched base repair and combining it with the induced mutagenesis model developed earlier. It was shown in detail which protein interactions play a role inátheámutual regulation of the two systems and how this regulation influences theáfinal yield of radiation-induced mutations. As a specific radiation type, ultraviolet was chosen because it allows one to exclude quite efficiently the influence of other systems whose functioning is observed under ionizing radiation or in the presence of some chemical agents. The research was performed by turning on or off specific proteins responsible for different stages of repair and, afterwards, comparing theámodeling results and experimental data. The model was used to study mutagenesis in bacterial cells with defects in the mutS, mutL, mutH, and umuC genes in different combinations. The results, which were based on a detailed model description ofátheámolecular mechanisms of the two systems, confirmed the hypothesis about the role of mismatched base repair in induced mutagenesis. Not only did theáperformed research allow a better understanding of the interconnection between theá two repair processes, which are different in nature, but it also cleared up the issue of what molecular mechanisms are behind the parameters of the classical exponential dependence describing radiation-induced mutagenesis in bacterial cells.
In parallel, primary interactions of heavy charged particles withá DNA were modeled. Work on the stated tasks led to the formation of a new field of research oná modeling DNA damage induction, where the spatial structure of the charged particle track andáŚ more precisely than beforeáŚ the geometry of the target are taken into account. At the first stage, model approaches were developed in which theáDNA damage induction mechanism is described in terms of the radial distribution of spatial energy and absorbed dose in a charged particle track. As an example, a comparison was performed of the spatial location of the atoms of an adenineľthymine nucleotide pair with a calculated radial distribution. It was established that the proposed approach would be more efficient with the use of tools for modeling the spatial structure of a charged particle track, which would allow describing theámechanisms of the induction of primary lesions of different types taking into account theáprecise atomic structure ofáDNA. In the studies to follow, it was necessary toátake into account theáinfluence of the mechanism of the bond break between DNA atoms onátheáspecifics of damage yield. Using the obtained results, it seemed possible to evaluate theáprobability of the induction of different types of lesions by heavy charged particles. Itárequired, however, complicated computational techniques connected with clearing up the quantum mechanical nature of DNA damage formation.
The stated task was solved by combining several model approaches. With theáuse of data on the DNA molecular structure, the exact spatial geometry of the linear section of the double spiral was modeled. Alongside with this, the use of transport codes TRIOL and GEANT4 allowed obtaining spatial models of tracks of heavy charged particles of different energies. The superposition of the geometric model of the target, which is a DNA section, and the spatial structure of the particle track yield information on the initial localization of energy deposition in the molecular structure of the double spiral. This made it possible to go over to the next stage: modeling the migration of the positive charges formed at the places of interaction between the particle track delta electrons and DNA atoms. With the use of the quantum mechanical apparatus, the probability of positive charge migration was evaluated, foráwhich several shortá Ś about 10 nucleotide pairsá Ś DNA sections had been chosen.
Theádependence of the shift of the chain bases on time was calculated. Itáwas shown that a deviation from equilibrium can point to a local destruction ofáthe chaináŚ that is, DNA damage of different types. Also, double spiral sections were identified, whereá DNA damage development is the most probable. In other words, theá proposed model approach allowed performing a probabilistic analysis of theá emergence ofábase lesions and DNA single- and double-strand breaks, which form heavy charged particle-induced clustered damage. Actually, this research cleared up the nature of energy fluctuations in sensitive microvolumes of cells, of which classics ofáquantitative radiobiology spoke.
Thus, since the beginning of research on the mathematical modeling of radiobiological effects, a wide range of issues have been considered and significant results have been obtained, which are topical and carry novelty. On these model approaches, hands-on classes were based that were offered at practical courses held at JINR for Russian and foreign young specialists. Materials of the research were included in graduate programs of Dubna University and Lomonosov Moscow State University.
The Molecular Dynamics Sector performed research on molecular dynamics modeling of radiation-induced conformation changes in protein structures and condensed matter (M. A. Ostrovsky, T. B. Feldman, Kh. T. Kholmurodov). The main obstacle on the way to the efficient MD application to extended macromolecules like proteins is well known. It consists in the huge size of the system (from tens of thousands to millions of atoms) and the time scale of calculating dynamic conformations of proteins (from femtoseconds to nanoseconds and longer). It is thus impossible toágo without powerful computational resources and the newest processors and platforms, which have to be adequately adapted to MD tasks. Finding relaxed conformation states of mutant proteins based on classical computational approaches can take years even for one protein structure.
The visual pigment rhodopsin is a typical representative of the large family ofáintegral membrane receptor proteins, which bind the G-protein (G-protein-coupled receptors, GPCR). These proteins play the key role in the information and regulatory processes in the organism. Calculations of rhodopsin molecular dynamics allowed finding out some special features of the conformational state of its chromophore: 11-cis-retinal. As is known, at least three types of opsin conformation states can be identified in the rhodopsin molecule: a)ádark-adapted, where the chromophore group (11-cis-retinal) functions as a powerful ligand antagonist preventing opsin interaction with the G-protein; b)á strongly activated light-adapted, where atáone of the final stages of photolysis Ś metarhodopsin II formation Ś all-transretinal functions as a powerful agonist efficiently facilitating opsin interaction with theáG-protein; c)áscantily activated light-adapted, where at the photolysis final stage opsin completely looses all-trans-retinal, and its chromophore place remains empty.
In a series of computer modeling studies, a comparative research was performed oná theá molecular dynamics of rhodopsin that contained the chromophore group (11-cis-retinal), free opsin (without 11-cis-retinal), and the rhodopsin mutant version associated with the development of the pigmental degeneration of the retina, which ultimately leads to complete blindness.
It is remarkable that in recent years only computer modeling based on X-ray structure analysis data allowed solving such problems. X-ray structure analysis yields a detailed three-dimensional static picture of the rhodopsin molecule iná itsá darkadapted crystal state. The molecular dynamics method allows describing the dynamics of the moleculeĺs conformational state, for example, chromophore and itsáinteraction with five surrounding amino acid residues or the dynamics of other domains of the molecule (like the cytoplasmic or intradisk ones). Besides, some nonvalence bonds (hydrogen, van der Waals, and electrostatic) in a crystallized protein molecule can be disordered due to the deformation of -spirals, which can play an important role in the functional properties of the visual pigment. Iná other words, theá X-ray structure analysis data cannot provide an unbiased three-dimensional picture ofátheámolecule. Moreover, the resolution of this method does not allow aádetailed description of chromophoreĺs three-dimensional ordering in theáprotein. Theoretical approaches can thus make it possible to describe the molecular dynamics at the atomic level and understand how the visual systemĺs unique ability to perceive a light quantum is realized.
Thus, the intramolecular mechanism of rhodopsin regeneration was disclosed.
During this process, the rhodopsin molecule acquires unique photochemical properties, which results in the visual pigment becoming a photoreceptor. Being atátheásame time a G-protein binding receptor, it is inactive in darkness and is practically incapable of interaction with the G-protein. Both of these functional properties of rhodopsin are of principal importance for the realization of phototransductionáŚ aánormal physiological process in the dark-adapted visual cell.
Another field of the Sectorĺs research is computer modeling of the CDC28 yeast kinase and homologous human CDK2 kinase. For yeasts, pleiotropic manifestations of mutations were shown to take place in the CDC28 gene. These mutations disturb the cell cycle, repair, and checkpoint control, thus leading to enhanced mutagenesis. For dynamic modeling, amino acid substitutions were used which have pleiotropic manifestations in the yeast cells cdc28-srm [Gly20Ser] and cdc28-13 [Arg283Gln]. The cdc28-13 mutation is localized in the large domain of the kinase and is remote from the site interacting with cyclin and ATP. The cdc28-srm mutation is a substitution for the third glycine in the conservative sequence GxGxxG inátheáso-called G-rich loop in the small domain of the kinase subunit; it is located against the T-loop in the large domain of the kinase subunit. It was found that the G- and T-loops areáimportant, but their specific role has not been cleared up yet.
Molecular dynamics modeling of the human CDK2 kinase (native protein: modeláI) was performed with substitutions for the respective amino acids CDK2-Gly16Ser (a mutant protein: model II) and CDK-7 Arg284Gln (a mutant protein: model III);
nanosecond dynamics ofá the CDK2/ATP complex was analyzed. The importance ofáthese amino acids was shown; their influence on CDK2 kinase conformation was clarified: itáisáindicated by an increase in the distance between the G- and T-loops in the respective mutant forms. The obtained results show that mutations destabilize the local structure in the region of the T-loop. The Arg-end region mutation has a more pronounced effect; itáleads to the loosening of the kinase structure and an increase ináthe distance between the G- and T-loops.
The Laboratory conducts radiation research (headed by G. N. Timoshenko)
ináthe following areas both of experimental and computational character:
Ľ participation in the development of JINRĺs new nuclear physics facilities asáregards the design and calculation of biological shielding, prediction of the radiation environment at specific facilities and their environment, evaluation of the induced radioactivity of the equipment, evaluation of the staff ĺs exposure, provision of radiation safety measures, and designing radiation monitoring systems;
Ľ verification of Monte Carlo methods of calculating radiation transport in matter by comparing the calculation results and experimental data or by using software with different intranuclear cascade models;
Ľ physics support of the LRBĺs research conducted at JINRĺs nuclear physics facilities; improvement of heavy nuclear beam dosimetry methods;
Ľ development of neutron spectroscopy methods in a wide energy range ináscattered radiation fields beyond the shielding of nuclear physics facilities; applied research using dosimeters based on solid-state detectors of damage marks and thermoluminescent detectors;
Ľ participation in the program of planet surface research with nuclear physics methods.
Following below are the 2005ľ2012 results in these fields of research.
In 2001ľ2005, LRB staff participated in designing the Subcritical Assembly ináDubna (SAD). In 2005, the work was mainly completed and the SAD project section ôRadiation Safety of the SAD Facilityö was prepared. The radiation environment was studied in detail on the territory around the Phasotron accelerator and the proton beam-based nuclear spectroscopy facility YASNAPP buildings (the Laboratory of Nuclear Problems) at different operation modes of the accelerator; the vertical distribution of the neutron dose rate over the Phasotron wall was measured inátheáregion of the vents and on the Phasotron roof; and the depth distributions of the soil radioactivity in the Phasotron levee were measured. The shielding calculations were done using the MCNPX radiation transport code.
To check the correctness of the calculation of the internuclear cascade that develops in the lead core of the subcritical assembly under exposure to 660-MeV protons from the Phasotron, an experiment was performed on measuring spectra of secondary neutrons from the target in the energy range of 50ákeVľ660áMeV at angles ofá45, 75, and 105 and angular distributions of hadrons by activation detectors with different energy thresholds. A comparison of this experimentĺs results witháMonte Carlo calculations using the MCNP4B + LAHET and MCNPX codes showed a good agreement between the calculated and experimental data.
As part of designing SAD shielding, a great amount of calculations of the radiation environment in the subcritical assembly building were done taking into account different radiation sources: 660-MeV proton beam losses in the beam transportation channel and in the magnetic optics elements; leakage neutrons from the assembly core shielding and from the Phasotron continuous shielding. On the basis ofá theá obtained data, the radiation exposure zones in the assembly building were determined for different operation modes; the neutron dose rate in the environment was calculated; and the activation of air and materials in the magnet room and soil underátheáfacility, as well as atmospheric emission activity, were evaluated.
In cooperation with the Laboratory of High Energy Physics, measurements were continued of spectra of neutrons generated byá 1 and 1.5á GeV protons inátheáU + Pb + CH2 assembly. The aim of the experiments at the Gamma-2 facility was to estimate the cross section of radioactive waste transmutation. AlsoáatátheáLHEPĺs request, the efficiency of proton beam transportation was studied and proportional ionization chambers were calibrated with the use of activation detectors.
From 2007, LRB staff members G. N. Timoshenko and M. Paraipan (Romania) participated in designing radiation shielding and developing radiation safety measures for the NICA complex. The requirements, information on radiation sources, the initial data for shielding calculations, and the tentative layout of the shieldings ofá theá booster, the Nuclotron, collider, and beam transportation channels made up theá contents of Sectioná 8 (Biological Shielding and Radiation Monitoring) ofáVolumeáIV of the NICA draft proposal (2009).
In forecasting the radiation environment at the NICA complex, of cardinal importance is the correct description of the sources of secondary radiation generated in matter by relativistic superheavy nuclei. For this purpose, Monte Carlo codes SHIELD, FLUKA, and GEANT4 for calculating radiation transport in matter were verified using unique experimental data on neutron yield from a thick iron target irradiated with 1áGeV/nucleon 238U nuclei. On the grounds of the verification results, GEANT4 was chosen as the basic code.
During work on the project, numerous versions of the complex concept, collider layout, and secondary radiation sources were considered; the criteria of radiation environment evaluation were changed accordingly. Different placements ofáthe collider canyon were examined: Building 205, a semiunderground construction, aáseparate building, etc.; different designs were proposed of the nuclear beam catchers, which are the main secondary radiation sources along the collider rings. For a work team of the Comet Close Corporation, source data for the shielding design were prepared. In particular, the double differential yield of neutrons and protons in the reaction 197Au + natFe at an energy of nuclei of 4.5áGeV/nucleon, spectral and angular distributions of hadrons from a thin target and a catcher, and neutron fluence and dose attenuation in concrete for the corresponding spectra were calculated. Also, otherá reference materials were provided. Agreement between the results obtained using the GEANT4 and SHIELD codes was good, which allowed the designers toáuse approximate engineering methods of shielding calculation for the NICA collider projectá(2011).
LRB staff participated in the development of the radiation safety measures atáall the projectĺs stages. Energy deposition in the superconducting coils of the magnetic dipoles and in the lenses was calculated for quenching probability evaluation;
theáshielding against bremsstrahlung from the colliderĺs system of electron cooling was calculated. A nontrivial problem of the activation of the collider rings by primary nuclei and secondary hadrons of the internuclear cascade was solved. The experimental data suitable for verifying induced radioactivity calculations are scarce, and the accuracy of simulating radionuclide production cross sections in nuclear reactions is poor. For this reason, a comparison was performed of partial activities in a thick iron and copper targets irradiated with a beam of 0.95áGeV/nucleon 238U nuclei as calculated using the GEANT4 and SHIELD codes. It was shown that the GEANT4 calculations of the total activity of the medium- and long-lived isotopes are acceptably reliable. The induced activity calculations based on the planned schedule of the colliderĺs 10-year operation allowed prediction of radiation environment dynamics inside the canyon when the collider is off and elaboration ofátheácriteria ofáclassifying the collider ring equipment as radioactive waste with reference toáspecific radionuclide activities.
A detailed 3D calculation of the colliderĺs radiation environment is being done using GEANT4. The errors of the engineering methods of shielding calculation and, if necessary, refinement at the detail planning stage are evaluated taking into account the shielding proposed by the designers.
The layout of the shielding against neutrons was proposed and calculated foráaástationary and mobile customs control facilities for detecting hidden drugs and explosives. A local shielding of two scrapers of the IREN facilityĺs electron accelerator was designed.
At the Nuclotron (the Laboratory of High Energy Physics), U-400M cyclotron (the Laboratory of Nuclear Reactions), and the medical beam of the Phasotron and Rocus-M therapeutic gamma-facility (the Laboratory of Nuclear Problems), radiobiological experiments were performed, in which biological objects were irradiated with particles of different physical characteristics: 170- and 1000-MeV protons;
1000áMeV/nucleon deuterons; 200, 500, and 1000áMeV/nucleon 4He, 12C, and 24Mg nuclei; low-energy 7Li, 11B, 14N, and 20Ne nuclei; and 60Co gamma rays. The irradiated objects included human peripheral blood lymphocytes, plant and organism cells, eye proteins, and small laboratory animals. Unfortunately, due to the long drawn-out upgrade of the Nuclotron, no radiobiological research was carried out atáits nuclear beams in 2007ľ2011.
For fast irradiation of a set of thin samples at the U-400M cyclotron, theáôGenomeö automated irradiation facility was used. In 2010ľ2011, it was completely upgraded. Itá was installed at a beam branch of the ACCULINNA fragment-separator and underwent first tests. For the calibration of its dosimetric ionization chamber, aáscintillation detector with an ultrafast analog-to-digital converter is used. Changes inátheáspectra of nuclear energy deposition in the detector allow beam quality control during sample irradiation.
There is no stationary irradiation facility at the Nuclotron, so it has to be assembled and calibrated before each experiment from the very outset. In addition, atá theá sample irradiation place (the F3 focus of the high-energy particle beam inátheáion guide gap), beam collimation is impossible; the experimental opportunities are thus limited. The short-term task of the LRBĺs experiments at the Nuclotron isátoáprovide a high quality of the heavy nuclear beam and precise low absorbed dose dosimetry for pulsed beam operation. For this purpose, it is necessary to carry out the analysis of the beam with respect to nuclear energy deposition in a thin detector, to ensure the precision measurement of the nuclear flux taking into account the time microstructure of the beam with the use of a telescope of scintillation counters, and toácalibrate the readings of the ionization chambers equipped with highly sensitive currentľfrequency transformers in a wide range of currents of different nuclear beams. The complete fulfillment of this task would be possible if there were a stationary irradiation chamber at a special medical and biological channel of the Nuclotron or booster.
The development of neutron spectrometry methods in a wide energy rangeáŚ from thermal to several hundred MeVáŚ for mixed and scattered radiation fields has been a top priority area of radiation research due to its practical importance.
Such a spectrometry is based on a multisphere methodology that uses the energy dependence of the neutron slowing-down length in a hydrogenous material. Overall, theámultisphere neutron spectrometer is a small slow neutron detector placed inside spherical polyethylene moderators of different diameters. The slow neutron detector options include 6Li-enriched LiI(Eu) scintillator, proportional spherical 3He counter, 198Au and 115I activation detectors, and a pair of 6LiF and 7LiF thermoluminescent detectors. The multisphere spectrometer is the only instrument to measure the neutron spectra (and, correspondingly, the dose) in the weak radiation fields beyond the accelerator shielding. The importance of the retention and development of this methodology consists also in that it is only the LRB and the Department ofáRadiation Research of the Institute of High-Energy Physics that have such 6LiI(Eu) crystalbased spectrometer and experience in operating it.
At the LRB, the multisphere technique developed towards the refinement ofá theá spectrometerĺs rated sensitivity functions, extension of the spectrum measurement energy range into high energies, and the creation of a portable version ofátheáspectrometer for field measurements. With the use of the MCNP code, precision calculations were performed of the spectrometer sensitivity functions for neutron energies upátoá20áMeV for monodirectional and isotropic radiation. An additional 10-inch polyethylene sphere with a lead insert 8ácm in diameter was manufactured for increasing spectrometer sensitivity at high energies.
In cooperation with Parsec Ltd., a portable stand-alone version of the multisphere spectrometer with a monitor was designed and manufactured. As the neutron field monitor, a proportional 3He neutron counter in a cylindrical polyethylene moderator is used with a charge-sensitive preamplifier. The spectrometerĺs scintillation sensor with a 6LiI(Eu) crystal is connected to the multichannel analyzer through a spectrometric analog-to-digital converter (ADC). The ADC, which has a large-capacity increment memory unit, is an external device hooking up to a USB port. The ADC memory unit is divided into two parts with independent inputs; so, in fact, there are two separate ADCs: for the spectrometer and monitor. The multichannel analyzer is based on the Lenovo S9 netbook. Another external USB-fed unit includes two high-voltage sources: for the photoelectronic multiplier of the spectrometerĺs sensor and for the 3He counter of the monitor. To extend the stand-alone operation ofátheámonitor-equipped spectrometer, an additional rechargeable lithium battery isá used. Theá battery has a USB port, to which the netbook or external units can be connected. To reduce the weight and size of the spectrometer for field measurements, an integrated multicomponent polyethylene moderator was made. It consists of concentric hemispheres; such a design allows a quick assembly of moderators ofáaárequired diameter. This multisphere spectrometer has a much lower total mass than the preceding version and can work for 12áhours in the mode of measuring neutron spectra without an external power supply. The Lenovo S9 netbook is powerful enough to run the Reconst software, which uses the statistical regularization method to solve the inverse problem, for the quick reconstruction of neutron spectra from spectrometer measurements done with different moderators.
Another experimental technique that was developed at the LRB for a long time was track detectors of damage traces. At JINRĺs accelerator beams, theásensitivities of polyalyl diglycol carbonate (PADC) and polyethylene terephthalate (PETF) detectors and LET dependences of track diameters for different nuclei were studied.
Fruitful cooperation was established between the LRB and the National Institute ofáRadiological Sciences in Chiba, Japan. Results of a comparison of different passive detectors used in space dosimetry were processed. The comparison was performed at 4He, 12C, 28Si, and 56Fe nuclear beams of the HIMAC medical accelerator. Ináaáresearch performed jointly with the Institute of Nuclear Physics in Prague, theáCzech Republic, CR-39 detectors were processed that had been irradiated inside the Russian module of the International Space Station (ISS) in 2005. Spatial distributions were obtained of the absorbed and equivalent galactic cosmic ray doses inside the module. The CR-39 detectors were used to study the fragmentation of high-energy 20Ne and 24Mg nuclei in light targets.
Within the framework of an interstate agreement on cooperation in science between Russia and India, LRB staff (V. E. Aleinikov) participated in a project oná theá synthesis of new nanocrystal thermoluminescent detectors for the dosimetry of heavy charged particles and electromagnetic radiation. The nanophosphors fabricated ináIndia were irradiated with 150-MeV protons at the Phasotron ofáthe Laboratory ofáNuclear Problems, 60Co gamma rays at sources in Dubna and New Delhi, and atáion beams of the Pelletron electrostatic accelerator ofátheáInterUniversity Accelerator Centre in New Delhi. Dependences of thermoluminescent detector sensitivity to protons, ions, and gamma rays on the absorbed dose were studied.
It was shown that with decreasing the thermoluminescent crystal size toá~ 10ánm, nanophosphors become more practical than microphosphors for theámeasurement ofáhigh doses of ionizing radiation.
The adequacy of indications of two gauges widely used at JINR to the ambient and individual dose equivalents was evaluated: the operational inspection neutron dosimeter based on the SNM-14 boron counter in a combined moderator and theáindustrial DVGN-01 personal albedo dosimeter. The study was a series of calculations using energy dependences of dosimeter sensitivities and neutron spectra ofáJINRĺs nuclear physics facilitiesáŚ both measured by LRB staff and taken fromátheáliterature. Gauge readings for fields with known spectra and radiation dose values for these spectra were calculated. With the use of the obtained results, radiation dose reading errors were determined, which allowed finding correction coefficients forádosimeter readings. 24áneutron spectra measured at JINRĺs basic nuclear physics facilities were used that correspond mainly to the neutron spectra at the most probable staff locations. The authors of this study won JINRĺs 2011 Second Prize foráApplied Research.
A technique was proposed for the approximate calculation of a comb-shaped filter used in targeted tumor therapy with carbon nuclei (M.
The Cosmic Gamma-Spectroscopy Laboratory of the Institute of Space Research (ISR), the Russian Academy of Sciences, is Russiaĺs planetology center. TheáLaboratory designed a number of instruments for planet surface studies by nuclear physics methods. Some of them were, and some are planned toábe, installed onáboard Russian and foreign spacecraft. Russian experiments are conducted oná board theá spacecraft of theá National Aeronautics and Space Administrationá (NASA), theáU.S., andátheáEuropean Space Agencyá(ESA) on theábasis of Intergovernmental Agreements between the Russian Federal Space Agency (Roscosmos) and theseáagencies. TheáLaboratoryĺs collaborators are a number of scientific organizations ináRussia, including JINR. The latterĺs tasks are the following: participation iná instrument development at the design stage; computational modeling of radiation environment atáplanetsĺ orbits and performances of instruments and their responses using universal codes of radiation transport in matter (MCNPX and SCINFUL-R); and the preparation and fulfillment of instrument calibration using radiation sources at modeling benches and in the field conditions.
Since the beginning of ISRľJINR cooperation in 1998, LRB staff (A. R. Krylov and G. N. Timoshenko) have participated in the evaluation of the characteristics and calibration of neutron detectors and gamma spectrometers for the following missions: the High Energy Neutron Detector (HEND) for 2001 Mars Odyssey, HEND Phobos for Phobos-Ground, the Lunar Exploration Neutron Detectorá (LEND) forá theá Lunar Reconnaissance Orbiter (LRO), the Dynamic Albedo of Neutrons (DAN) complex for the Mars Science Laboratory (MSL), the on-board neutron telescope BTN-Neutron for the International Space Station (ISS), and the Mercurian Gamma and Neutron Spectrometer (MGNS) for BepiColombo.
The HEND instrument on board the 2001 Mars Odyssey orbiter was the first toáshow that there are huge subsurface water ice deposits in the polar and even middle latitudes of the Mars, which was a very significant scientific result.
The LEND instrument was installed at NASAĺs LRO, which was launched iná theá summer of 2009. LEND was intended for studying the chemical composition of the lunar ground; it was the first high spatial resolution neutron telescope in space research history. Its main task was the search for water in the lunar ground or onátheásurface. The instrument worked efficiently; two LRB staff members wereá awardedá NASAĺs letter of commendation for the successful realization ofátheámission.
New instruments were designed for the evaluation of the chemical composition of the Marsĺs moon Phobos (HEND Phobos for Roscosmosĺs Phobos-Ground mission) and the Mercuryĺs surface (MGNS for ESAĺs BepiColombo mission, which is planned to be launched in 2015). Besides neutron detectors, these instruments include LaBr3 scintillator-based high-resolution gamma spectrometer.
The DAN complex was created for NASAĺs MSL Curiosity rover. The mission isáaimed at the evaluation of water content in the Martian ground on the roverĺs path with a horizontal resolution of about 1ám at depths of up to 1.5ám. The instrument isásuccessfully performing on the Martian surface near Gale Crater.
On board the service module of the Russian segment of ISS, the high-energy neutron telescope for the BTN-Neutron experiment has been operating sinceá2006.
The aims of this experiment include research on secondary neutron radiation inátheáEarthĺs upper atmosphere generated by high-energy charged particles, theáneutron component of solar flares, and the neutron component of the radiation background on board ISS.
New-generation instruments (ADRON-LR) are being designed for the evaluation of the elemental composition of the Moonĺs surface at the spacecraft landing site by active neutron and gamma spectroscopy. The spacecraft are planned toábeálaunched in 2015 and 2017.
Experimental work was performed at JINRĺs EG-5 electrostatic generator toá calibrate instruments and study their physical characteristics using 252Cf and 239PuľBe radioisotope neutron sources and 0.2ľ15.3áMeV monoenergetic neutrons fromátheáreactions p + 7Li = n + 7Be; d (D, n) 3He; and T( d, n)4He. Modified 252Cf
-based neutron sources in spherical polyethylene moderators 3 and 5 inches in diameter were also used.
To calibrate the energy scales of the instrument spectra of the pulses of the stilbene-based high-energy neutron detector and LaBr3 crystal-based gamma detector, gamma rays from isotope sources and neutron capture and inelastic neutron scattering reactions on iron, nickel, and nitrogen were used. The measurements were carried out both using isotope neutron sources and at a thermal neutron beam ofátheáIBR-2 reactor.
In model experiments, the water or ice-containing ground layer was simulated by a calcium silicate brick assembly with a polyethylene layer at different depths ofáup toá1ám. Also, full-scale tests were conducted on a concrete-covered flat open surface with ice-simulating polyethylene layers. For model tests with a neutron generator, a special bench was made that will allow simulation of different compositions of the Martian ground with great variability.
In 2013, the Astrobiology Sector was established at the LRB. It has been headed by A. Yu. Rozanov, Academician of the Russian Academy of Sciences. The scope ofá theá Laboratoryĺs new unit includes biogeochemical studies of cosmic matter onátheáEarth and in nearby space and research on the biological and geochemical specifics of the early Earth. The main objects of research are the cosmic materials that are parts of meteorite composition, micron-sized cosmic dust particles, and rocks and fossil organisms of the early Earth. The Sector is active in the following
Ľ biogeochemical studies of cosmic dust;
Ľ studies of biofossils and organic compounds in meteorites and in ancient terrestrial rocks;
Ľ research on the synthesis of prebiotic compounds from formamide under exposure to space types of radiationáŚ on the Earth and in space.
The first area involves studying cosmic dust (CD) in different terrestrial terrains and CD collection in the upper atmosphere and in nearby space. CD research allows evaluating the regularities in the time distribution of cosmic dust falling onátheáEarthĺs surface, which is important for the reconstruction of the geological history of the Earth and obtaining data on the paleoclimate. Studying the CD structure, mineralogical, elemental, and isotope composition, and biological properties will help solving fundamentals problems like the nature of interplanetary matter and its role in the origináof life.
In the course of the work in this field, CD samples were collected in different terrestrial terrains (Arctic and Antarctic snow and ice, high mountain snow and ice, peat moss, rock strata, bottom sediments, the upper atmosphere, near-Earth space, and interplanetary space); isolation (enrichment) of the space component of the collected dust samples was performed. A comprehensive analysis of the space
component of dust has been underway, which includes:
Ľ studying the mineralogical, chemical, and elemental composition of CD;
Ľ determination of the isotope composition of CD reference elements;
Ľ search for biomarkers in CD: biofossils, organic compounds, metabolites, nucleic acids, and viable cells;
Ľ estimation of the total amount of CD falling on the Earth surface;
Ľ evaluation of the CD spatial distribution over the the Earth surface and its temporal variations; studying CD composition variations in the Earthĺs geological history;
Ľ comparative analysis of fossil CD and interplanetary CD collected byáspacecraft.
Biofossils and organic compounds in meteorites and ancient terrestrial rocks are another field of the Astrobiology Sectorĺs research. Biofossils are petrified microorganisms and products of their vital activity. They are an important tool for studying bacterial life occurrence. Studying biofossils in meteorites and ancient terrestrial rocks allows obtaining data on the forms of ancient terrestrial and extraterrestrial life and clearing up the problem of the origin of life. An important discovery was made by scientists of the Paleontological Institute of the Russian Academy of Sciences: microorganism traces were found in meteorites. It is remarkable that some ofátheámeteorite rocks with bacterial life traces are older than the Earth. It is a strong reason toásuggest that life on the Earth is not unique: somewhere in the Solar system (orábeyond) it had emerged earlier than the Earth formed. Within this field of research, the
Sector performs the following specific activities:
Ľ selection of samples of metasedimentary, volcanogenic sedimentary, and volcanogenic rocks of the greenstone belts of Karelia and Kola Peninsula;
Ľ preparation of samples for scanning electron microscope studies;
Ľ scanning electron microscopy of the samples.
An important field of the Sectorĺs research is studying the regularities and mechanisms of the formation of prebiotic compounds from formamide (NH2COH)áŚ one of the simplest chemical compounds abundant in the interstellar and interplanetary medium. In cooperation with specialists of Italian universities (Prof.á R. Saladino ofá Tuscia University, Viterbo, and Prof.á E. Di Mauro of La Sapienza University, Rome), experiments are conducted in which samples of different meteorites, mixed with formamide, are exposed to ionizing radiation (high-energy protons and heavy ions). In these conditions, production of nucleic bases, carboxylic acids, amino acids, sugars, and other complicated compoundsáŚ up to nucleosidesáŚ was observed.
Experiments are planned on the synthesis of nucleotides from these products.
The elemental composition of the meteorites used in this research is determined by neutron activation analysis, which is performed jointly by the LRB Astrobiology Sector and the Laboratory of Neutron Physics at the IBR-2 pulsed reactor (M. V. Frontasyeva).
INTERNATIONAL COOPERATIONFrom the first steps towards the establishment of the Biological Research Sector (BRS) at the Laboratory of Nuclear Problems (LNP) in 1978, JINRĺs radiobiologists began an active cooperation with specialists of JINR Member States.
Among the radiobiologists who participated in research conducted by the BRS was aáteam ofáscientists of Berlin-Buch Institute. The team was headed by Prof.áH. Abel andáDr.áG. Erzgreber. JINRĺs cooperation with Berlin-Buch Institute grew from contacts between radiobiologists of this institute and the Institute of Medical Radiology iná Obninsk, where in the 1960sľ70s the world-renowned geneticist and radiobiologist Prof.á N. V. Timofeev-Ressovsky worked. Under the influence of his studies, aná actively working school of radiobiologists was formed in Berlin-Buch before the Second World War. Therefore, after the establishment of the BRS, which wasáthen headed byáProf.áV. I. Korogodin, who had earlier worked for many years with N. V. Timofeev-Ressovsky, JINR started its own collaboration with German scientists.
The research conducted jointly with Berlin-Buch Institute was focused on theámolecular mechanisms of DNA lesions in higher organism cells induced by accelerated heavy ions. During a short time, a set of equipment was created, which made it possible to study regularities and mechanisms of DNA double-strand breaksá(DSBs) inámammalian cells cultivated in vitro. Unique results were obtained, which allowed finding out different aspects of the lethal effect of radiations of different physical characteristics on higher organism cells.
At the same period, the BRS collaborated with the Institute of Nuclear Chemistry and Technology (Warsaw, Poland). On the Polish side, this work was headed byáDr.áO. Rosek. The research was aimed at making a comparative study ofátheálethal effect of radiations in a wide range of linear energy transferá(LET) on two lymphoma cell lines that had different repairability ofáDNA damage. It was shown that there isáaásignificant difference between the radiosensitivity of these two cell lines: theáradioresistant one had a normal repairability ofáDNA damage; the radiosensitive one had a defect in the repair system. With an increase in heavy charged particleáLET, theá radiosensitivity of both cell lines was observed to level off, which pointed toátheáinduction of direct DNA DSBs by high-LET radiations.
The cytological effect of ionizing radiations on plant cells cultivated iná vitro was studied at the BRS byáE. Hlinkova of the Comenius University (Bratislava, then Czechoslovakia). In the early 1980s, F. Czaba, a mathematician at the Central Institute foráPhysics (Budapest, Hungary), did at the BRS theoretical research onámodeling the spontaneous mutation process in lower eukaryote cells. At the same time, V. Lisy, a theoretician at the University of Koice (then Czechoslovakia), studied at theáBRS the problem of the presence of Davydov solitons in DNA.
In the early 1980s, the BRS began the active development of radiobiological research at the accelerators of the Laboratory of Nuclear Reactions, JINR. The main aim was to find out the mechanisms determining the differences in the biological effectiveness of ionizing radiations of different physical characteristics. This work was joined by two scientists of then Czechoslovakia: S. Kozubek (the Institute ofáBiophysics, Czechoslovak Academy of Sciences, Brno) and, later, V. Michalik (the Institute of Radiation Dosimetry (IRD), Prague).
S. Kozubek developed a model describing the regularities in the lethal effect ofáradiations in a wide LET range on bacterial cells with different repairability ofáDNA damage. This model allowed the description of the lethal radiation effects in bacterial cells (the cell survival shape, radiosensitivity dependence on LET, oxygen effect, and effect of radioprotectors of different classes) induced by heavy charged particles.
It was shown that the specifics of the effect of multicharged ions on the genetic apparatus of cells can be determined by the cluster type of the DNA damage induced byáheavy ions.
A microdosimetry analysis of the yield of DNA lesions of different types under ionizing radiations of different physical characteristics was performed byá V. Michaliká (IRD). It was shown that with increasing LET, the yield of cluster lesions of single- and double-strand DNA increases. This dependence is described byá aá curve with a local maximum, the maximum location being different for different types of cluster lesions. It was a pioneering study, which was later continued inámany research centers of the West.
A wide range of research was conducted in 1985ľ1990 to study the mutagenic effect of radiations of different LET on cells by an international team of physicists and radiobiologists, which included M. Bonev (the Institute of Nuclear Physics and Nuclear Energy, Bulgaria), S. Kozubek (then Czechoslovakia), B. Tokarova (thená Czechoslovakia), and F. Czaba (Hungary). To find out the relative role ofátheáphysical and biological factors in the induced mutation process, S. Kozubek initiated research on the induction of forward and reverse mutations in bacterial cells.
It was found that the dose dependence of the cell mutation frequency has aálinear quadratic character. It was shown that with an increase in particle LET, theácharacter of the mutation frequency dependence on the irradiation dose does not change; it is only the relative genetic efficiency (RGE) of radiations that changes. TheáRGE dependence on LET is described by a curve with a local maximum. Within theáframework of the theoretical approach developed by S. Kozubek, the difference was explained ináthe locations of the maximums of the relative biological effectiveness dependence on LET against the lethal and mutagenic effect criteria. It is determined by the different character of the DNA lesions participating in mutagenesis and theálethal effects of radiation. The former are mainly damaged bases; the latter, DNA DSBs. Iná1989, S. Kozubek successfully defended a doctoral thesis on this subject.
M. Bonev studied in detail the regularities and mechanisms of prophage lambda induction by radiations of different physical characteristics. This research allowed an evaluation of the role of the inducible repair system in prokaryote cells during theámutation process caused by ionizing radiations of different quality.
Since 1985, effective cooperation has been going on with a group of GSI radiobiologists (Darmstadt, Germany) headed by Profs.áG. Kraft and S. Ritter. Specialists ofá theá Department of Radiation and Radiobiological Research (DRRR) and theirá German colleagues have been performing experiments at heavy ion beams ofá the GSI accelerator to study the cytogenetic effect of accelerated heavy ions oná mammalian cells iná culture and human lymphocytes. DRRR specialists made aánotable contribution to the preclinical investigations of the radiobiological characteristics of multicharged ion beams designed for cancer therapy.
In 1990ľ1998, close collaboration was maintained with the Radiation Biology Division, the Institute of Aerospace Medicine (Cologne, Germany), of the German Aerospace Center. On the German side, a team headed by Dr.áG. Horneck participated in this research, which was concerned with the development of a new method ofá studying the kinetics of the expression of the inducible cell operons based onátheáluciferase reaction. The international team developed an efficient and simple method (SOS-Lux test), which allowed a real-time evaluation of damage in the genetic apparatus of living cells under ionizing radiation, ultraviolet light, and chemical carcinogens. For this purpose, a genetic construction was assembled which contained genes of luminescent bacteria controlling the synthesis of proteins involved in the luminescence reaction (lux genes). When DNA is damaged, gene functioning repression isá removed, which triggers the luminescence reaction. As a result, theá cells carrying the mentioned genetic construction emit visible light; the light yield depends directly on the degree of DNA damage and can be easily measured.
Thus, theáSOS-Lux test, in its essence, proved to be a unique biological dosimeter;
itácan be widely used in different areas: in ecologyáŚ for the rapid analysis of chemical carcinogen and mutagen contaminations; in pharmacologyá Ś for evaluating theápossible mutagenicity of new medicines; in chemical and food industry.
To advance these promising studies, the team got financial support inátheáform ofáa grant from the Copernicus program (Brussels, Belgium). The research resulted in the creation of a device which allows on-line detection of physical and chemical mutagenic factors in the environment.
In the genetics of yeast cells, joint work was performed during several years witháProf.áN. Babudri of the University of Perugia (Italy) to investigate the genetic control of mutagenesis under cell starvation. This task was related to the problem ofá theá genetic control of cell cycle arrest after DNA damage induction. In recent years, the interrelation has been more evident between different components ofátheáintegral cell response to DNA damage, which stands behind genome stability and integrity. A relation is shown between the cell cycle control mechanisms and DNA repair mechanisms. The mechanism of the control and coordination ofáthese processes was discovered in the late 1980s; it was called checkpoint control.
Thisámechanism allows the cells to survive and maintain genetic stability and is regulated by the checkpoint genes. It is believed that a disorder in checkpoint ways that leads to an increase in mutability and genome instability is important in the early stages of carcinogenesis.
In 1988ľ1997, DRRR radiobiologists productively collaborated with theáNational Aeronautics and Space Administration (NASA) of the U.S. On theá NASA side, this work was headed by Dr.á T. Young. Within the framework of the Agreement ofáCooperation between JINR and NASA, a series of experiments were performed atá the JINR Synchrophasotron to determine the relative biological effectivenessá (RBE) ofá 1ľ5á GeV protons. In experiments on human blood lymphocytes, regularities ofáthe induction of stable and unstable chromosome aberrations were studied. Theá RBE values of relativistic protons were found to be not higher than those ofágamma radiation.
The DRRR has been actively cooperating with the Institute of Biophysics ofátheáAcademy of Sciences of the Czech Republic (the city of Brno) to study theácytogenetic mechanisms of the induction of stable chromosome aberrations in human cells by radiations in a wide LET range. On the Czech side, the collaboration isá headed byá Prof.á S. Kozubek. Joint research on the cytological effect of heavy charged particles on plant cells has been conducted with specialists of Comenius University (Bratislava, Slovakia). Fruitful cooperation has been developing with the Institute ofá Nuclear Chemistry and Technology (Warsaw, Poland). This work, which, onátheáPolish side, is headed by Prof.áA. Wojcik, is focused on studying regularities and mechanisms of the induction of different types of chromosome aberrations (unstable chromosome lesions and translocations) by different doses of accelerated charged particles. Also, the DRRR regularly performs similar research jointly withá theá GSI (Darmstadt, Germany). Active cooperation has been formed with Minsk State University (Belarus): the joint work is aimed at studying the cataractogenic mechanisms of high-energy heavy charged particles and mechanisms ofátheáeffect ofáradiations of different quality on the visual pigment rhodopsin.
The uniqueness of JINRĺs nuclear physics facilities and their ionizing radiation fields required the development and production of new means of radiometry and ionizing radiation dosimetry. In the 1960s, M. Zielczynski, a JINR scientist from Poland, created a recombination dosimeter of mixed ionizing radiation, which allowed measuring absorbed and equivalent doses and radiation quality coefficients for beams and scattered radiation fields of the accelerators and pulsed fast neutron reactor.
Information on the energy dependences of dosimeter sensitivity is essential forá measuring the characteristics of ionizing radiation fields that are complicated ináterms of their component make-up and energy distribution. For this reason, studying the performances of the dosimeters and detectors that are in use iná theá JINR Member States has been one of the main fields of international cooperation over the past decades. The energy dependences of the sensitivity of a Bonner spectrometer, solid state and emulsion track detectors, and thermoluminescent detectors were studied in cooperation with many specialists of these countries, including M. Gelev and I. Mishev (Sofia, Bulgaria); B. Derschel, G. Hahn, H. Taut, L. Wetzel etá al.
(Dresden, Germany); S. Pszon and M. Zielczynski (Swierk, Poland); D. Nikodemova and M. Fulop (Bratislava, Slovakia); F. Spurny, Z. Spurny etá al. (Prague, the Czech Republic); and others.
To evaluate the accuracy of measuring the characteristics of radiation fields byáinstruments that are in use in the JINR Member States, a series of comparative measurements were made in the 1970s in the fields of JINR proton accelerators, ináaábeam of the IBR-30 reactor, in 252Cf-based fields in polyethylene moderators, and in the fields of CERN accelerators. These measurements were performed by specialists of Bulgaria, Czechoslovakia, Poland, Romania, and the Soviet Union. Theáresults showed that the required accuracy of measuring the characteristics of radiation fields can only be achieved occasionally. This research resulted in improving the dosimetry techniques used in the JINR Member States.
After DRRR establishment, the cooperation between JINR and theáInternational Atomic Energy Agencyá(IAEA) was broadened. This cooperation included theáfollowing fields: specific research at the request of the IAEA; JINRĺs participation inátheáIAEA-coordinated research programs; and the organization and conduction ofátheáIAEA training courses.
The IAEA, in particular, exercises control over nuclear nonproliferation. In this regard, measuring weak neutron fluxes in intense gamma radiation fields to track the movement of fissile materials is an important task. To an order by the IAEA, performances were studied of different thermal neutron detectors with polyethylene moderators, equipment parameters were optimized, and a corona counter-based prototype neutron monitor for detecting the movements of nuclear materials was manufactured and tested in intense gamma fields.
The monitoring of staff irradiation using personal dosimeters is a significant component of evaluating the efficiency of any radiation safety program aimed atálimiting occupational exposure. A change in the radiation safety concept that followed the publication of new recommendations by the International Commission on Radiological Protection (1990) encouraged the development of new international radiation safety standards, which were prepared by international organizations, approved by the IAEA Board of Directors, and published in 1996.
These standards introduced new operational quantities for radiation monitoring. In particular, the standards direct that a new operational quantity be used forá the personal dosimetry of strongly penetrating radiation: the Hp(10) personal dose equivalent, which ensures that the requirement not to exceed the established dose limits is met. Taking into account the technical difficulties of introducing new radiation quantities for the measurements of radiation doses, the IAEA launched aáresearch program to compare the personal dosimeters used in its member countries ofáEastern Europe. The aim of the program was to enable the personal dosimetry services to evaluate the energy and angular dependences of the sensitivity ofádosimeters and to measure the radiation field characteristics in terms of the Hp(10) personal dose equivalent.
At the request of the IAEA, due to the DRRRĺs great experience in studying theáperformances of personal dosimeters and capabilities of providing metrological support for dosimetry measurements, JINR took part in this program as a metrology laboratory. Within the framework of this activity, the readiness was checked ofá23ápersonal dosimetry services for measuring Hp(10) in gamma radiation fields with different energy distributions of particles; also, the energy and angular sensitivity functions of the dosimeters in use were measured in terms of the personal dose equivalent. The performed research led to a significant improvement of the reliability of the measurements of the personal dose equivalent made in the IAIA member countries of Eastern Europe.
In 1996 and 1999, the IAEAĺs training courses for young specialists were conducted on the basis of the JINR University Center. The courses were attended byáseveral tens of specialists of most of the JINR Member States and Estonia, Latvia, and Lithuania.
EDUCATIONFor about 25 years, programs of training specialists in radiobiology, as well asá iná protection physics and dosimetry, have been offered at JINR. At the early stages of the formation of the JINR University Center (UC), the Department ofá Radiobiology was established there as a branch of the respective Department ofá Moscow Engineering Physics Institute (MEPI); also, postgraduate programs iná the specialty of Radiobiology were started. The Departmentĺs enrollment was made up of physics students who had completed seven semesters at MEPI, Moscow State University, Moscow Institute of Physics and Technology, and a number of other institutions ofáhigher education. After thesis defense, many of them were enrolled inápostgraduate programs and defended their Candidateĺs theses.
In 1998, on the initiative of the JINR Directorate, the Department of Biophysics was established at Dubna University. The Department trains certified specialists in the field of Human and Environmental Radiation Safety with the following specializations: Radiation Biophysics and The Biophysics of Photobiological Processes.
TheáDepartment provides mathematical, physical, chemical, and biological education through the following general and special courses: general biology; molecular biology; general radiobiology; clinical radiobiology; physiology; cytology; microbiology; biophysics; biochemistry; DNA damage and repair; radiation genetics;
theáphotochemistry and photobiology of the primary processes of vision; radiation protection; radiation dosimetry; mathematical modeling in radiation physics, biology, and ecology; and some others. The Department conducts research in radiobiology, radiation genetics, the kinetics of the primary photobiological processes, cytology, molecular biology, medical uses of radionuclides, microdosimetry, and mathematical modeling of dynamic biological processes. To consolidate theoretical knowledge, practical training is provided at the JINR-based student laboratories ofámicrobiology, cytology, molecular biology, photobiology, radiation dosimetry and protection, and experimental methods of nuclear physics. The Department offers postgraduate programs in the specialty of Radiobiology.
The Departmentĺs faculty consists of top-level specialists, including Prof.áM. A. Ostrovsky, an Academician of the Russian Academy ofáSciencesá(RAS);
professors and scientists of JINR, MEPI, the Institute of General Geneticsá (RAS), and other major scientific research centers. The Department is headed byá Prof.á E. A. Krasavin, Dr.á Biol. Many of the LRBĺs present-day young specialists graduated fromátheáDepartment of Biophysics.
CONCLUDING REMARKSThis publication presents a wide range of radiobiological studies conducted at the Laboratory of Radiation Biology of the Joint Institute for Nuclear Research (LRBáJINR). The diversity of the Instituteĺs sources of ionizing radiations with different physical characteristicsáŚ first of all, heavy charged particle acceleratorsáŚ predetermined the Laboratoryĺs main radiobiological research area: the regularities and mechanisms of the biological action of ionizing radiations in a wide range of linear energy transfer. The use of heavy ion accelerators allowed solving one of radiobiologyĺs key problems: the problem of the relative biological effectiveness of ionizing radiations. As was said in the first part of this book, the first radiobiological experiments at JINR were conducted more than 60áyears ago by specialists of the Institute of Industrial Hygiene and Occupational Diseases, Oncological Research Center, and a number of other Moscow institutes. In 1963, after the establishment of the Institute of Biomedical Problems (IBMP) of the USSR Ministry of Health, its specialists started experiments at JINRĺs accelerators to study the regularities and mechanisms of the biological action of high-energy protons. This research was necessary because highenergy protons are present in space and are very dangerous for cosmonautsĺ health.
The proton energy range of the Synchrocyclotron (JINRĺs first accelerator) allowed modeling the biological action of high-energy protons of space origin. With the assistance of the JINR DirectorateáŚ first of all, Director ofátheáLaboratory of Nuclear Problems (LNP) V. P. Dzhelepov, a Corresponding Member ofáthe USSR Academy of SciencesáŚ IBMP staff members performed a huge amount of radiobiological research on the relative biological effectiveness of high-energy protons. Then, at a lowenergy heavy ion accelerator of the Laboratory of Nuclear Reactionsá(LNR), research was started on the biological effects of accelerated multicharged ions. This work received attention and active support from LNR Director Acad.áG. N. Flerov and, later, Acad.áYu. Ts. Oganessian. These studies were focused on the specifics ofáthe action of densely ionizing radiations on living cells of different types: microorganisms (yeast and bacterial cells), experimental animal tissues (cornea and skin), and vegetable objects. Important results were obtained onátheálethal action of heavy ions on cells and development of chromosome lesions in irradiated cells. After the establishment in 1978 of the Biological Research Sectorá (BRS) atá JINR, which was actively supported by LNP Director V. P. Dzhelepov, cooperation with the IBMP rose toáaánew level. Some of its staff members went over to work at the BRS. Thus, theácontinuity of the traditional ties between the IBMP and JINR was provided. Space radiobiology has always been present in the fields of research performed by JINRĺs radiobiologists.
Currently, in connection with ambitious ideas of developing the Moon and ofátheáMars mission, it has become especially urgent to combine radiobiological research efforts because preparation of such flights requires providing protection from galactic space radiation. It seems that the microgravity factor will not be the main obstacle forátheálong-term flights beyond the Earthĺs magnetosphere since many issues ofátheáorganismĺs exposure to weightlessness have already been successfully resolved. Onátheácontrary, full protection from galactic radiation is still impossible in deep space. During a one-year flight there, 1ácm2 of a cosmonautĺs body will be hit by up to 105 heavy charged particles of the carbon and iron groups. The biological effectiveness of the iron group nuclei is very high. They are very likely to induce mutations and cancer; they induce cataract and are also harmful for the retina; and they affect the central nervous system cells.
Such exposures can be modeled at charged particle acceleratorsáŚ in particular, at JINRĺs Nuclotron. In modeling the biological effects of heavy charged particles at new-generation accelerators, to the fore comes research on the molecular mechanisms of genetic apparatus damage, which can allow evaluation of the character ofáthe induced DNA structure disorders, because the damage induced by galactic radiation is qualitatively and quantitatively different from the damage induced byáelectromagnetic radiations. One has to know exactly what mutations emerge and how dangerous they are; to estimate the probability of cancer and cataract development; and to estimate the risk of brain damage, because exposure to heavy charged particles can impair important functions of the central nervous system. Solving all these problems requires fundamental radiobiological research at high-energy accelerators ofámulticharged ions. Heavy charged particles are also a unique tool that allows yielding much knowledge on the organization of living systems. Heavy charged particle radiobiology is a new radiation biology; it is different from the classical one, which is concerned with the action of electromagnetic types of radiation. Efficient radiobiological research methods are being developed; thus, there are grounds toáexpect that not only a number of applied problems (in particular, theáones connected with planning the Mars mission), but also the fundamental problems ofáradiation genetics as part of life sciences will be solved.
The LRBĺs prospective research fields are defined by the Laboratoryĺs cooperation with the Department of Physiology of the Russian Academy of Sciences. Theávisiting session of the Departmentĺs Bureau held in the summer of 2013 in Dubna gave the LRB a strong impulse for further development as a research center. The sessionĺs aim was to work out a new concept of the radiation safety of interplanetary flights.
Itáwas proposed that new approaches to the solution of the radiation barrier problem be elaborated and that the possibility in principle of interplanetary flights be evaluated for the current level of space engineering. NASA, ESA, and other space agencies follow the established approaches, estimating the risk associated with radiation exposure as the probability of the development, first of all, of cancer as a delayed consequence. The LRB has developed new approaches to the solution of this problem.
The present-day radiation risk concept for orbital and interplanetary flights isá based on a generalized dosimetric functional as the criterion and quantitative measure of radiation danger. The generalized dose comprises the radiation doses inducing immediate and delayed effects. The immediate radiation-induced effects appear during the flight; the delayed ones develop throughout life. When the dose isácalculated for the immediate and delayed effects, coefficients are introduced taking into account the influence of radiation quality on the radiobiological effect (where radiation includes heavy charged particles of different energies), dose distribution over time, dose distribution over the human body, and modification of the organismĺs radiation response caused by other space flight factors. But this approach isáunacceptable for interplanetary flights because the action of heavy nuclei differs from that of electromagnetic radiations: a single particle track can be likened to a ôbullet pass,ö where huge amount of local energy is released, while the latter, to an electromagnetic ôrain.ö The action of heavy charged particles on the brain structures atádoses corresponding to real heavy particle fluxes for the Mars mission causes marked disorders of spatial orientation and cognitive functions. It was established that heavy ions damage hippocampus Ś the most important brain structure responsible foráworking memory formation. In this part of the central nervous system, neurogenesis goes onácontinuously Ś the production of new neurons involved inátheáformation ofáworking and long-term memory. The latest results obtained byáneurophysiologists suggest that the radiation risk concepts for interplanetary flight crews developed earlier have to be revised. Studying the neurophysiological effects of space radiations isá becoming one of radiobiologyĺs main tasks Ś interesting and complicated, requiring the conduction of research ranging from genetic structure damage to higher behavioral functions.
The first steps in this direction were taken at the LRB in 2013, when rhesus monkeys (offered by the IBMP) were irradiated at the 170-MeV proton therapy beam ofátheáPhasotron (the Laboratory of Nuclear Problems) and at the 500áMeV/nucleon carbon nuclear beam of the Nuclotron (the Laboratory of High Energy Physics)áŚ three animals at each beam. The brain was irradiated at an absorbed dose ofá1áGy.
Before the experiment, the animals were trained to solve test problems at a computer.
The aim of the experiment was to evaluate the disorders of the acquired skills caused by brain exposure to heavy charged particles with relatively low linear energy transfer. After irradiation, the animals were returned to the IBMP for further research.
Huge amount of experimental work has to be done at accelerators since there are a lot of unresolved issues. In particular, it is necessary to study the damageability of cells of the genetic structures controlling the synthesis of the proteins which participate in the functioning of the membrane channels and provide interaction between neurons in the synapses. The LRBĺs radiobiologists urgently need a broad international collaboration with neurobiologists. Solving the stated super-problem in Russia alone would be extremely difficult.
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First radiobiological experiments at JINR
Establishment of the Biological Research Sector
Development of the Biophysics Department at the Laboratory of Nuclear Problems
JINRĺs Department of Radiation and Radiobiological Research................122 Laboratory of Radiation Biology
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Ë˝Ű. ´ň¸. Ű. 14,75. Ë¸.-Ŕšń. Ű. 17,47. ĎŔÓŠ 435 řŕš. ăÓŕÓš 58550 ╚šńÓ˛ňŰŘ˝ŕŔÚ ţ˛ńňŰ ╬ß˙ňńŔÝňÝÝţŃţ ŔÝ˝˛Ŕ˛ˇ˛Ó ńňÝű§ Ŕ˝˝ŰňńţÔÓÝŔÚ 141980, Ń. ─ˇßÝÓ ╠ţ˝ŕţÔ˝ŕţÚ ţßŰÓ˝˛Ŕ, ˇŰ. ĂţŰŔţ-╩■Ŕ, 6 E-mail: firstname.lastname@example.org www.jinr.ru/publish/