[Influence of indoinetophene on the antiradiation properties of indralin].

Radioprotective properties of indralin were studied at its combined administration with indometophene in the periods optimal for each preparation before acute radiation exposure. Animals were subjected to total radiation on the IGUR installation (137Cs): mice of the strain (CBA x C57B1) F1 at a dose of 9 Gy (LD100/30), purebred dogs--4 Gy (LD100/45). It was established in the experiments on mice that considerable radioprotective effect can be obtained by the use of indralin at a dose that is half the optimal radioprotective dose if it is applied against the background of indometophene administered at its optimal radioprotective dose four days before. The survival of mice increased on the average by 30-35% and provided the same effect of protection as a single indralin at the optimal radioprotective dose (100 mg/kg). The survivability of dogs after the combined application of the two radioprotectors makes up 43% against 14% after application of only indralin at a dose of 5 mg/kg (half the optimal radioprotective dose). Indometophene, along with strengthening the antiradiation activity of indralin at the ineffective (half the optimal) dose, allows the reduction of its undesirable postradiation effects in the hemopoietic tissue. The important role in the mechanism of the antiradiation activity of indometophene and indralin belongs to the increased ribonucleotide reductase activity and induction of the ribonucleotide synthesis that provides effective reparation of the damage to the DNA of the cells in radiosensitive tissues and organs as a result of administration of protective doses of radioprotectors at the optimal doses before radiation exposure.

[Recombinant thrombopoietin antiradiation therapeutic effectiveness evaluation on dogs according to hemopoiesis and survival criteria].

Recombinant human thrombopoietin (rh TPO) has been investigated as a means of acute radiation disease urgent treatment in the experiments on 24 mongrel dogs. The animals were exposed to total acute gamma-irradiation at the doses of 3.5 Gy (exceeding LD50/45 under our conditions) and 3 Gy. All the dogs including control ones received antibiotics Ampicillin and Gentamicin twice a day during the acute period from the 7th to the 21st day. TPO was injected one time s/c or i/v at the doses of5 or 10 mkg/kg 1.5-2 h after exposure. TPO at a dose of 5 mkg/kg was ineffective. TPO at a dose of 10 mkg/kg had a positive effect on the kinetics of blood forming units, especially platelets (nadir, restoration rate) in terms of the 45-day survival. As a result, in TPO groups, nadir averaged at both exposure doses on leucocytes (1.3-1.4) x 10(9)/l vs (0.70-0.75) x 10(9)/l in control groups and on thrombocytes (102-112) x 10(9)/l vs (44-33) x 10(9)/l in control ones. Despite the low number of animals in experimental groups, the results permit to regard rhTPO as a worth-while urgent therapeutic means for the acute radiation damage treatment and preventing thrombopenia.

[Evaluation of the treatment effectiveness of domestic G-SCF preparations in experiments on irradiated dogs].

We have evaluated the treatment effectiveness of Leucostim and Neupomax in dogs exposed to radiation at lethal doses of 3 and 3.5 Gy, correspondingly, by testing the dynamics of the blood cell number, first of all, leucocytes and neutrophiles, and the 45-day survival. Supportive therapy for all the dogs, including the control ones, consisted in antibiotic treatment during the acute period of 7-24 days. It was shown that both pre-parations administered consecutively for about 17-21 days after irradiation positively influenced the dynamics of all blood cells but predominantly impacted the neutrophile number dynamics. The latter ones manifested a higher nadir level and an earlier onset of restoration in the G-SCF treated dogs in comparison with the control ones. The tendency to a positive influence on the survival has been shown in Neupomax-treated dogs exposed to 3.5 Gy of radiation (plus about 40%). The results of the experiments were in good accordance with the data by foreign authors who used Neupogen. This allows a conclusion that home-produced G-SCF preparations can replace their foreign analogues.

[The use of EPR spectroscopy to control the changes in organism radioresistance. Clinical results].

It has been shown that changes in Fe(3+)-transferrin and Cu(2+)-ceruloplasmin pools, which are trust-worthy controlled by the EPR technique in whole blood, blood plasma, and serum, as well as changes in the extracellular DNA content in blood plasma are markers of changes in organism radioresistance. This has been proved during the medical examination of the Chernobyl accident recovery workers and civil population, including children, exposed to low-intensity radiation, as well as during clinical investigation of new radioprotectors.

[The use of EPR spectroscopy to control the changes in organism radioresistance. Experimental results].

The responses of deoxyribonucleotide (dNTP), DNA, and protein synthesis systems in blood-forming organs of animals (dogs, mice) as well as changes in Fe(3+)-transferrin (Fe(3+)-TF) and Cu(2+)-ceruloplasmin (Cu(2+)-CP) pools in blood to gamma-irradiation and the administration of radioprotectors have been studied. It has been shown that changes in Fe(3+)-TF and Cu(2+)-CP pools in blood are indices of changes of body radioresistance and are reliably controlled by the EPR technique. An increase in the Fe(3+)-TF pool promotes the activation of synthesis of dNTP, DNA, and Fe(3+)-containing proteins, which are essential for repair efficiency during early post-irradiation time as well as for the development of compensatory and restorative reactions of cellular systems; i.e., they are responsible for body resistance to DNA-damaging factors. It is important that the intensity of responses depends on the initial state of the organism. Thus, dogs with initial individual characteristics of blood typical for "suppressed" or "activated" states had abnormally high responses to irradiation by low doses of 0.25 and 0.5 Gy. This fact is important for the estimation of consequences of prolonged low-dose irradiation for human population. It has been shown that radioprotectors, efficient in survival test activate the synthesis of dNTP, DNA, and proteins in organs. The intensity of dNTP synthesis and the time when dNTP pools get maximum values determine the efficiency of protectors and the time of irradiation after their administration.

[Activation of deoxyribonucleotide synthesis by radioprotectants and antioxidants as a key stage in formation of body resistance to DNA-damaging factors].

The responses of the systems of synthesis of deoxyribonucleotides (dNTPs), DNA, and proteins in hematopoietic organs and liver of animals to gamma-radiation, administration of radioprotectants and antioxidants as well as the dependence of these responses on the doses of radiation and drugs were studied. Radioprotectants of acute (indralin) and durable effects (indomethaphen) as well as natural (alpha2-tocopherol) and synthetic anti-oxidants (ionol or 2,6-di-tert-butyl-4-methylphenol) efficient in survival test were used. Three stages could be recognized in the standard unspecific response of the studied systems to radiation: (1) immediate increase in ribonucleotide reductase activity in the tissues within the first 30 min as a part of the integrated SOS response to DNA damage, which activates dNTP synthesis; (2) inhibition of the synthesis of dNTPs, DNA, and and (3) restoring ribonucleotide reductase activity and integral increase in the production of dNTPs, DNA, and total protein, which is essential for the development of compensatory and restorative responses of the organism. The radioprotectants significantly increased ribonucleotide reductase activity, which increased intracellular concentrations of the four dNTP types in organs during radiation exposure and three following days. Within this period, ribonucleotide reductase activity was inhibited by 40-50% in animals not treated with radioprotectants as compared to control. Balanced high pools of dNTPs in the organs of radioprotectant-treated animals provided for high-performance repair of DNA damage. The radioprotectant-induced activation of dNTP synthesis during the development of compensatory and restorative responses provides for an earlier restoration of the cellular composition and functioning of the organs. Antioxidants stimulated the synthesis of dNTPs, DNA, and proteins in animal tissues in a strict dose interval. Their effect on the studied syntheses was dose-dependent: single or multiple long-term administration of high antioxidant doses inhibited synthesis of dNTPs, DNA, and proteins. Radioprotectants and antioxidants affected the pool of blood protein Fe3+-transferrin controlling the synthesis of iron-containing ribonucleotide reductase activity in hematopoietic organs, and hence, the iron-dependent stage in DNA synthesis--dNTP synthesis. Activation of protein synthesis in organs by the studied substances increased the pools of Fe3+-transferrin and Cu2+-ceruloplasmin in the blood, which activated dNTP and DNA synthesis. Activated synthesis of dNTP, DNA, and proteins in the organs and increased pools of studied plasma proteins underlay the formation of body resistance to DNA-damaging factors.

[Reactions of deoxyribonucleotide synthesis system to irradiation and their modification by radioprotectors]. / Reaktsii sistemy sinteza dezoksiribonukleotidov na obluchenie i ikh modifikatsiia radioprotektorami.

The paper covers the problem on reactions of deoxyribonucleotide (dNTP) synthesis system in blood-forming organs of animals induced by irradiation. The synthesis of dNTP is a rate-limiting stage for DNA synthesis. Cellular requirements for dNTP pools during DNA synthesis are related with ensuring of the accuracy of DNA copying during replication and repair. It has been shown that organism defence mechanisms against irradiation include the following stages: 1. The prompt SOS-activation of dNTP synthesis 30 min later after irradiation, playing the important role in protecting of cell's genetic apparatus from damage. 2. The inhibition of dNTP synthesis within 3-24 h after irradiation resulting to the imbalance of four dNTP and the decrease of their pools. As result of that, the abnormal repair is observed due to depurinations, errors of base incorporations and "misrepair". 3. The restore of dNTP synthesis occurred 2 days later after irradiation. The increase of dNTP pools promotes the increase of DNA synthesis rate as well as proliferative activity of cells. Confirming the fact that the alterations in dNTP pools play essential role in the production of DNA lesions became an important step in understanding of the multistage process leading to radioprotection. To get high and balanced pools of dNTP needed for the increase in the volume of repair of DNA lesions the radioprotectors with high efficiency relative to the survival test were used in experiments. They induced the elevated dNTP synthesis in bone marrow and spleen during the time when the irradiation alone caused the essential prolonged suppression of dNTP synthesis as well as DNA and protein synthesis in organs of nonprotected animals. It has been shown that substances with antioxidant and antiradical activity induced the dNTP synthesis, too. In vivo regulatory factors of dNTP synthesis have been studied to elucidate the mechanisms of getting of high and balanced dNTP pools by using of different substances.

[Time- and dose-dependent post-irradiation changes of Fe3+-transferrin and Cu2+-ceruloplasmin pools in blood, their influence on ribonucleotide reductase activity in animal tissues and the effects of radioprotectors]. / Vremennye i dozozavisimye postradiatsionnye izmeneniia v soderzhanii Fe3+-transferrina i Cu2+ tseruloplazmina v krovi zhivotnykh i ikh vliianie na ribonukleotidreduktaznuiu aktivnost' tkanei.

The time- and dose-dependent changes of Fe(3+)-transferrin (Fe(3+)-TF) and Cu(2+)-ceruloplasmin (Cu(2+)-CP) pools, of superoxide dismutase activity and the inhibitory activity of alpha 2-macroglobulin in blood as well as changes in synthesis rates of deoxyribonucleotides (dNTP), DNA and proteins in organs (spleen, liver, bone marrow, thymus) of mice and dogs given total body irradiation have been studied using of ESR spectroscopy, radioisotope techniques and biochemical determination of enzymatic activity. The experimental data have allowed us to reveal the sequence of organism's response reactions against irradiation and their modifications by radioprotectors. Changes in blood Fe(3+)-TF pool is one of the most informative, highly radiosensitive and rapidly reactive marker against irradiation and drug administrations. This irontransport protein controls a rate-limiting iron-dependent stage for DNA synthesis--the synthesis of dNTP, catalyzed by iron-containing ribonucleotide reductase (Fe(3+)-RR). It has been shown that time-dependent post-irradiation changes of Fe(3+)-TP pool in blood are characterized by three distinct stages: 1) the prompt increase of pool (SOS-type response) playing the important role in protecting of cell's genetic apparatus from damage; 2) the decrease of its pool within 3-18 h after irradiation resulting in the loss of Fe(3+)-RR activity in tissues of blood-forming organs that make more stronger radiation-induced damage; 3) the following phase-dependent increase in Fe(3+)-TF pool at the 2-nd, 6th, 10-17th days after irradiation due to an increase in transferrin synthesis. This increase may be considered as compensatory reaction of blood-forming organs directed at restoring blood and organ's cells. The time-dependent courses of the reactions are independent from radiation doses indicating to the universal and nonspecific response of organism against irradiation. But, the intensity of this compensatory-adaptive response at 2-nd and 6th days grows with increasing radiation dose up to lethal that, and organism's response becomes abnormal and physiologically hypertrophic. The prolonged "stressful syndrome of biochemical tense state" should be attributed to negative effects for organism, since it may result in the failure of compensatory adaptive organism's reactions and animal killing. The radioprotectors ward off the appearance of this dangerous state. Dogs with initial individual characteristics of blood which were typical for "suppressed" or "activated" states had abnormal response against irradiation by low doses 0.25 or 0.5 Gy. In these cases the intensity of response reactions of organism was essentially increased and markedly deviated from linear dose dependence. The phase-dependent increase of Fe(3+)-TF pool in blood in post-irradiation time resulted to the increase of Fe(3+)-RR activity in blood-forming organs. The key event ensuring the development of compensatory adaptive reactions is the increase of capacity of protein-synthesizing apparatus, the activation of biosynthesis of dNTP and DNA against the treatment with damaging factors.

[Influence of the indometafen radioprotector on the immune reactivity]. / Vliianie radioprotektora indometofena na immunologicheskuiu reaktivnost' organizma.

In the experiments on dogs and mice it was shown that indometophene could modify the immune status of the body. Specific and non-specific resistance of mice to E. coli infection increased after indometophene administration. At the same time higher titers of normal anti-salmonella antibodies in mice serum and a higher phagocytic activity of neutrophils in the peripheral blood in dogs were found. The influence of the preparation on the content of the complement and normal antibodies-hemagglutinins was less pronounced. It was suggested that the indometophene ability to bustle immune reactivity of the body played a certain role in the mechanism of its radioprotective effect.

[Indometofen causes biochemical changes in the blood cells characteristic for a radioresistant state of the body]. / Indometofen vyzyvaet v kletkakh krovi biokhimicheskie izmeneniia, kharakternye dlia radiorezistentnogo sostoianiia organizma.

The DNA and RNA contents, RNA/DNA ratio, and spontaneous and latex-induced oxidant activity indices of the whole blood were studied in the nitroblue tetrazolium test of mono- and polymorphonuclear blood leucocytes of intact dogs after injection of lipopolysaccharide pyrogenal. Significant changes in the above parameters were revealed for radioresistant (survived) and radiosensitive (lost) animals exposed to a subsequent prolonged gamma irradiation with a lethal dose of 7.64 Gy (LD75/45). Peroral introduction of 30 mg/kg indometofen (an indole analog of tamoxifen), which is a potential radioprotector, to dogs increased the survival rates of the irradiated dogs up to 93% and aided in the adaptive biochemical changes in the nuclear cell compartment of blood to induce a radioresistant status of the organism.

[The molecular aspects of the action of the radioprotector indralin]. / Molekuliarnye aspekty deistviia radioprotektora indralina.

The effect of indralin on the metabolic parameters in peripheral blood and organs of irradiated dogs and mice have been studied by EPR, NMR and radioisotope methods. It has been shown that indralin stimulated biosynthesis of DNA precursors as well as of DNA and proteins in the organs and stabilized the rate of ATP and glycogen synthesis. As a result indralin reduced considerably the changes produced by gamma-irradiation on the macromolecular biosynthesis during the early post-irradiation period. Indralin has induced marked favorable changes in the rate of macromolecular synthesis, normalized the ATP and glycogen content, induced ribonucleotide reductase activity and increased the Fe(3+)-transferrin content during development of compensatory-repair response in the irradiated animals. Indralin prevented hyperdevelopment of the repair response and its breakdown due to radiation-induced exhaustion of viability of many important cellular and body systems after irradiation with lethal doses.

[The molecular mechanisms of the action of the radioprotector indometafen. The biosynthetic and bioenergetic aspects]. / Molekuliarnye mekhanizmy deistviia radioprotektora indometafena. Biosinteticheskie i bioénergeticheskie aspekty.

It was shown that indomethaphen (IM) is capable of stimulation of the synthesis of DNA, RNA, and protein precursors in mice. The IM-induced elevated level of the ribonucleotide reductase activity and, hence, deoxyribonucleotide pool in the spleen at the moment of irradiation and during the early postradiation period provides for complete DNA repair. As a result, the damaging effect of ionizing irradiation is weakened. At later stages (2-20 days) IM activates protein and DNA synthesis leading to the recovery of the ribonucleotide reductase activity in the spleen, on increased content of Fe3(+)-transferrin, cytochrome-c-oxidase, and ferrosulfuric components of the mitochondrial electron transport chain, and increased potential of the detoxication system due to the elevated content of cytochrome P-450. IM stimulates ATP synthesis. Thus, IM enhances compensatory-restorative reactions of the cell systems, more pronounced in the spleen than in the liver.

[The dose dependence of the development of compensatory-restorative body reactions to irradiation. The EPR method]. / Dozovaia zavisimost' razvitiia kompensatorno-vosstanovitel'nykh reaktsii organizma pri obluchenii. Metod EPR.

The study deals with the mechanism of organism's adaptive responses to the effect of radiation in widely ranging dose. Post-irradiation metabolic changes were evaluated in canine blood as well as in murine blood, spleen, bone marrow and liver using the EPR spectroscopy. It was shown that the dynamics of changes in transferrin and ceruloplasmin pools and ribonucleotide reductase activity were phase-dependent with the maxima at the 2nd, 6th and 10-12th days after irradiation. Such dynamics was observed at various irradiation doses applied. The data allow us to suggest that the nonspecific compensatory--adaptive reactions of organisms develop as the response to irradiation. The dose-response function of the reaction intensity was found to be linear. The shape of the dose-response curve indicates that the minimum response of organism depends on the dose linearly up to 3.2 Gy (for dogs) as well as the maximum one. However, in the case of low-dose irradiation (0.25 or 0.5 Gy) there were deviations of maximum responses from the linearity, i.e. the amplification of the amplitude of compensatory adaptive reactions. These effect were shown to be dependent upon initial individual characteristics of animal blood and to be related to the "depressed" or "activated" state of organism prior to irradiation. The ribonucleotide reductase activity was measured in bone marrow and spleen of animals by the EPR method. The nature of non-repairable DNA damage is discussed in view of the inactivation of ribonucleotide reductase.