[The most important stages of the mechanism of action in vivo of carcinogen diethylnitrosoamine and its effect on the synthesis of RNA, proteins, DNA, and deoxyribonucleotides].

The mechanisms of nitric oxide (NO) generation from exogenous and endogenous sources, induced by the addition of the carcinogen diethylnitrosoamine (DENA) to rat organism have been studied. Within 15 h after the addition of DENA, the carcinogen itselt acts as an exogenous NO donor. The products of protein degradation (the process induced by DENA) act as endogenous donors of NO. It was shown that the generation of nitric oxide from diethylnitrosoamine leads to deep hemic and tissue hypoxia and induces the inactivation of oxygen-dependent enzymes, including ribonucleotide reductase, and the inhibition of ATP synthesis. Under these conditions, the protein synthesis and as a consequence the synthesis of deoxyribonucleotides and DNA are strongly suppressed; i.e., diethylnitrosoamine produces the effect similar to the action of the antibiotic cycloheximide, an inhibitor of translation. The administration of cycloheximide to the animal organism also led to the appearance of a considerable amount of nitric oxide in the blood. It is assumed that nitric oxide initiates (on the administration of the carcinogen) or at least enhances (on the administration of cycloheximide) the blockage of the synthesis of the protein, deoxyribonucleotides, and DNA. In response to the disturbance of protein synthesis, the complex of enzymes is activated that accomplish the utilization of the degradation products of proteins, including the inducible form of NO synthase.

[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.

[The inhibitor of free radical processes decrease of protein biosynthesis in gun short wound tissues and weaken development of the general adaptation syndrome].

The dynamics of total protein biosynthesis and procollagen biosynthesis in skeletal muscle of injury tissues with the antioxidant BHT (dibunol) treatment and with common healing were studied. The obtained date indicate that the AO treatment reduce the rate of biosynthesis both the total proteins and procollagen at the 3th day of healing. Dibunol also considerably reduce the protein biosynthesis in adrenals and brake of corticosteroids biogenesis as measured by ESR-signals intensity of reduced adrenodoxine. AO treatment also reduce the protein biosynthesis in thymus, spleen and bone marrow. The lowering of functional activity of endocrine and immune systems indicate that the AO significantly inhibit the systemic reactions of organism induced by acute wound affect. It was suggested that as "primary mediator" of stress-reaction may be considered lipoperoxide radicals and decay products of lipohydroperoide.

[The mechanisms of nitric oxide production from exogenous and endogenous NO-donating compounds and its effect on deoxyribonucleotide and DNA synthesis].

The mechanisms of exogenous nitric oxide (NO) production through in vivo biotransformation of nitro-, nitroso- and amino-containing substances were discussed. In addition, the mechanisms of production and cellular sources of endogenous NO, appearing in the blood and tissues after the exposure to various DNA-damaging factors, have been considered. Considerable quantities of endogenous NO were detected in the body in the first hours after translation inhibition by cycloheximide or animal exposure to superlethal radiation doses, i.e., after the exposure to factors inducing destructive processes. The time and dose dependences of exogenous and endogenous NO production have been established. NO produced after a single or repeated administration of NO-donating compounds as well as endogenous NO proved to inhibit deoxyribonucleotide (dNTP) and DNA synthesis in animal tissues. Nonspecific compensatory responses to disturbed protein homeostasis included cyclic production of endogenous NO. The maximum levels of nitrosyl complexes were registered when the rate of protein synthesis decreased. The role of polyamines in the induction of macromolecule biosynthesis is discussed and NO production from these arginine-rich compounds is proposed. NO is released at the stage of polyamine inactivation. The inactivation mechanism includes the hydroxylation of aminogroups by NO synthase, the formation of nitroso intermediates, and their denitrosation with NO release.

[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.

[Anabolic effect of natural and synthetic antioxidants]. / Anabolicheskii éffekt prirodnykh i sinteticheskikh antioksidantov.

The order of responses of cell systems of organs and changes in the content of some proteins of mouse and dog blood in response to addition of natural (alpha-tocopherol) and synthetic (ionol) antioxidants was studied at the whole-body level using ERP spectroscopy, radioisotope analysis, and chemiluminescence technique. Responses were evaluated by the temporary and concentration-dependence changes in the activity of ribonucleotide reductase and the rate of protein and DNA synthesis in organs of mice, as well as by the changes in the pools of Fe3+ -transferrin and Cu2+ -ceruloplasmin in blood and the antiradical activity of blood plasma of dogs and mice. During the first 24 h of exposure to alpha-tocopherol, the activity ribonucleotide reductase in bone marrow rapidly increased, whereas the activity of this enzyme and the rate of DNA synthesis in the thymus and spleen were suppressed by 30-50% compared to the control. The changes in these parameters had a phase mode with maxima on days 2-3 and 6-8. The stimulatory effect of the antioxidant on the processes of synthesis was concentration-dependent. We found that the optimal stimulation of the synthesis of deoxyribonucleotides, DNA, and protein was achieved by single administration of alpha-tocopherol at a dose of 20 mg per dog with an average weight of 15 kg and 17 mg/kg in the case of mice. Single or repetitive administration of higher doses of alpha-tocopherol was either ineffective or even suppressed the synthesis of DNA and deoxyribonucleotides. Ionol administered at a dose of 60 mg/kg increased DNA and protein synthesis in mouse organs in 2-4 and 1.2-1.5 times, respectively, compared to the control. It was also shown that single and repetitive administration of alpha-tocopherol to dogs increased the pool of Fe3+ -transferrin and Cu2+ -ceruloplasmin in blood in 2-3 times and by 20-30%, respectively, compared to the control. It is suggested that changes in Fe3+ -transferrin pool in peripheral blood may be used for evaluation of the stimulatory effect of antioxidants on the synthesis of macromolecules in organs and for the determination of dependence of this effect on the concentration of antioxidants.

[Reaction of rats organ cells to inhibition of protein biosynthesis by sublethal doses of cycloheximide]. / Reaktsii kletok organov krys na ingibirovanie biosinteza belka tsiklogeksimidom v subletal'noi doze.

Time-dependent responses of cellular systems in rat organs and Fe(3+)-transferrin and Cu(2+)-ceruloplasmin pools in blood to the blocking of translation by sublethal doses of cycloheximide (CHI) was studied by EPR spectroscopy and radioisotope techniques. It was shown that, within the early post-CHI-treatment time, the suppression of deoxyribonucleotide and DNA biosynthesis, the activation of catabolic enzymes, the inhibition of electron transfer in the mitochondrial electron transport chain, the activation and the following inactivation of cytochrome P-450, and an intensive production of nitrosyl complexes in rat blood and organs occur. In addition, the activation of the synthesis of steroid hormones in adrenal gland was revealed within 1-24 h after cycloheximide injection. In response to these metabolic disturbances, nonspecific compensatory recovery reactions developed, first of all, the "reprograming" of the translation process to produce new protein-synthesizing elements instead of cycloheximide-blocked ones. The activation of protein synthesis promotes the recovery of deoxyribonucleotide and DNA synthesis, the restoration of the redox state of mitochondrial and microsomal electron transport chains in organs as well as an increase of Fe(3+)-transferrin and Cu(2+)-ceruloplasmin pools in rat blood. These metabolic processes result in the full recovery of the functional ability of organs.

[Ecdysterone modulates antitumor activity of cytostatics and biosynthesis of macromolecules in tumor-bearing animals]. / Ekdisteron moduliruet protivoopukholevuiu aktivnost' tsitostatikov i ikh deistvie na biosintez makromolekul v organakh zhivotnykh-opukholenositelei.

The influence of therapeutic and half doses of cisplatin and adriamicin combination with the anabolic drug ecdisteron (20-hydroecdison) on development of subcutaneously and intraperitonially transplanted P388 and L1210 leukemia and metastasizing B16 melanoma was studied. Ecdisteron significantly stimulated the chemotherapeutic effect of low doses of the cytostatics: inhibition of tumor growth, mice survival rate, their lifespan, and the antimetastatic activity index were comparable or better than after therapy with high doses of the antitumor drugs. The influence of high and low doses of cisplatin and its low dose in combination with ecdisteron on the dynamics of protein and DNA biosynthesis in the liver, pancreas, thymus, spleen, and adrenals of tumor-bearing mice were also studied. Although the therapeutic effect of 4 mg/kg cisplatin by activated protein biosynthesis and DNA repair is comparable or better than that of its low dose (2 mg/kg) in combination with ecdisteron, in terms of chemotherapy the combination looks preferable since the therapeutic dose of cisplatin is toxic for the intact tissues.

[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 structural characteristics of the polyribosomes in the rat liver studied by means of a tritium label]. / Osobennosti struktury poliribosom pecheni krys, izuchennye metodom tritievoi metki.

Polyribosomes isolated from the rat liver in a medium with low ionic strength were irradiated by "hot" tritium atoms under conditions providing for the replacement of the hydrogen atoms located at the surface of polyribosomes by tritium. After fractionation of such polyribosomes, the radioactivity of the obtained fractions was measured and their proportions were calculated for the total surface accessible for the tritium atoms (in %), as well as their specific radioactivity. The material loosely associated with the polyribosomes and containing amino acyl-tRNA-synthetases is more radioactive than rRNA and r-proteins, especially concerning their specific radioactivity. This suggests that the material is organized as individual molecules located on the surface of ribosomes. The specific radioactivity of the RNA-component of this material (tRNA) is twice that of proteins, thus suggesting its surface localization in the composition of loosely associated material. Based on the pattern of labeling of rRNA and r-proteins of the native and preliminarily dissociated polyribosomes, we propose that the material, loosely associated with the polyribosomes, has affinity to both rRNA and r-proteins.

[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.

[Polyribosomes, isolated from rat liver in a low ionic strength medium, capable of autonomic translation in a cell-free system without the addition of cellular fluid]. / Poliribosomy, izolirovannykh iz pecheni krys v srede s nizkoi ionnoi siloi, sposobny k avtonomnoi transliatsii v beskletochnoi sisteme bez dobavleniia kletochnogo soka.

Polyribosomes isolated from the liver in the presence of 10 mM KCl and purified by centrifugation through 2 M sucrose were shown to incorporate [3H]leucine both into aminoacyl-tRNA and polypeptides in a cell-free system without cell sap. The incorporation of [3H]leucine showed a linear increase within 80-100 min and was then levelled off. The system was sensitive to cycloheximide, puromycin and ethionine and needed ATP, GTP and unlabeled amino acids. The quantitation of tRNA in polyribosomes (the fraction which did not sediment with the subparticles after polyribosome dissociation) revealed more than two tRNA molecules per 80S monosome. It is likely that this tRNA excess as well as the earlier established presence of aminoacyl-tRNA synthetases and elongation factors promote the autonomic translation of polyribosomes.

[Dynamics of the biosynthesis of components of the protein synthesizing apparatus of the rat liver at the stage of restoration of translation, inhibited by cycloheximide]. / Dinamika biosinteza komponentov beloksinteziruiushchego apparata pecheni krys na étape vosstanovleniia transliatsii, ingibirovannoi tsiklogeksimidom.

The biosynthesis of proteins, ribosomal RNA and other components of the rat liver protein-synthesizing system during the reparation and subsequent activation of translation inhibited by a sublethal dose cycloheximide (CHI, 3 mg/kg) was studied. It was found that the incorporation of labeled precursors into proteins and ribosomal rRNA isolated from free and membrane-bound polysomes is repaired already 3 hours after CHI injection. 6-9 hours thereafter, the level of component labeling reaches control values, whereas the total protein biosynthesis is retarded. After 12-24 hours, marked stimulation of ribosome biosynthesis and the integration of ribosomes into polysomes are observed together with an asymmetric accumulation of excessive amounts of newly synthesized 40S subunits into polysomes 12 hours after CHI infection. The putative mechanisms of the activation of expression of the part of the genome responsible for protein and ribosomal rRNA synthesis as well as for the synthesis of other components of the protein-synthesizing system are discussed.

[Rat liver energetics according to data of 31P-NMR in extreme states of the processes of biosynthesis of proteins and nucleic acids]. / Energetika pecheni krysy po dannym 31P-IaMR pri ékstremal'nykh sostoianiiakh protsessov biosinteza belkov i nukleinovykh kislot.

The dynamics of phosphomonoesters, phosphodiesters, Pi, ATP, ADP, NAD(H+) and uridine diphosphoglucose (UDPG) levels in rat liver upon sharp oscillations in the rates of protein and nucleic acid biosynthesis induced by a sublethal++ dose of cycloheximide was studied, using the 31P-NMR method. The results obtained with preparations of native liver are unaffected by fractionation, homogenization and chemical extraction procedures. It was demonstrated that oscillations of Pi, ATP and UDPG levels in liver cells reflect the changes in the energy consumption and intracellular energy-linked processes (e.g., glycolysis, oxidative phosphorylation, glycogen synthesis and consumption) under conditions of variable macromolecular synthesis rates. The oscillations in phosphomonoesters and phosphodiesters levels are mainly due to cycloheximide-induced lipid metabolism disturbances.

[Characteristics of [3H]cycloheximide distribution, protein and DNA biosynthesis in rat organs after sublethal blocking of translation]. / Osobennosti raspredeleniia [3H]tsiklogeksimida, biosintez belka i DNK v organakh krysy dosle subletal'nogo bloka transliatsii.

The kinetics of accumulation and release of [3H]cycloheximide (CHI) as well as protein and DNA biosyntheses in some organs of the rats injected with sublethal doses of CHI were studied. It was shown that in the majority of organs under study (especially in the liver, kidneys and adrenals) the inhibition is completed within 12 hours after CHI injection followed by the resumption of protein and DNA syntheses. In the thymus and pancreas the levels of these biosyntheses remain below control values up to the 72nd hour. A positive correlation was observed between the decrease of CHI (or its metabolites) concentration and the beginning of protein and DNA syntheses in different organs. However, there was a reverse correlation between the high values of squares below the kinetic curves of CHI release from the liver, kidneys and adrenals and the intensive resumption of protein and DNA biosyntheses in these organs. It was thus assumed that in these particular organs CHI is subjected to intensive biotransformations. The contribution of the endocrine system to the induction of intensive compensatory protein and DNA syntheses in the liver were estimated from the viewpoint of the nature of reconstructive processes occurring in the appropriate organs.

[The 31P-NMR study of the metabolism of phosphate-containing compounds in the rat liver during inhibition of protein synthesis]. / Izuchenie metabolizma fosfatsoderzgashchikh soedinenii v pecheni krysy metodom 31P-IaMR v usloviiakh podavleniia sinteza belka.

Using the 31P-NMR method, the composition of the pool of phosphate-containing metabolites in intact rat liver 72 hours following the blocking of protein biosynthesis by cycloheximide was studied. It was shown that during maximal inhibition, i.e., 2-3 hours after cycloheximide injection, the ATP concentration decreases approximately 5-fold, that of ADP and sugar phosphates--4- and 2-fold, respectively. The intracellular pH in hepatocytes was followed by measuring the chemical shift of the Pi signal. The reconstitution of intracellular pH after 2-3 hours is consistent with changes in the Pi level in hepatocytes. The experimental results were compared with the data of biochemical analysis. NMR seems to be a promising tool in the study of metabolism of various animal organs and tissues under physiological and pathological conditions.

[The effect of gamma-hydroxybutyric acid on the biosynthesis of macromolecules and metabolic paramagnetic centers in hepatocytes in acute radiation injury]. / Vliianie gamma-oksimaslianoi kisloty na biosintez makromolekul i metabolicheskie paramagnitnye tsentry v gepatotsitakh pri ostrom luchevom porazhenii organizma.

It has been shown, that the single injection of gamma-hydroxybutyric acid (GHBA) (100 mg/kg) to mice 30 min before irradiation (6 Gy) prevents whole-body irradiation-induced inhibition of DNA at early post-irradiation period. GHBA stimulates the biosynthesis of macromolecules at 1-2 days after irradiation. GHBA also prevents the increase in the degree of reduction of the mitochondrial and microsomal electron transport chains at early post-irradiation time (up to 2 h.), that takes place only under irradiation. It means, that GHBA inhibits the production of O2 radicals, which induce lipid peroxidation processes at post-irradiation period.

[Radiation-induced free radicals in DNA, DNA level in tissues and tissue radiosensitivity]. / Radiatsionno-indutsirovannye svobodnye radikaly DNK, soderzhanie DNK v tkaniakh i tkanevaia radiochuvstvitel'nost'.

Radiochemical yields (G-values) of H-adducts of thymine bases of DNA (TH) in frozen (77 K) gamma-irradiated mouse and rat tissues were measured. The content of DNA and the number of TH radicals formed in DNA mass of 10(12)D at a dose of 1 Gy (beta parameter) were determined for each of the studied tissue. It was shown that beta parameter, which indicated DNA in situ radiosensitivity, was different for different tissues: it was higher for radiosensitive tissues. The possible causes of the effect observed are discussed.

[Activation of nuclear polypeptide synthesis in rat liver cells after inhibition of template protein synthesis. The role of nuclear polypeptide synthesis in the changes of the chromatin structure]. / Aktivatsiia iadernogo polipeptidnogo sinteza v kletkakh pecheni krys pri tormozhenii matrichnogo sinteza belkov. Vklad iadernogo polipeptidnogo sinteza v izmenenie struktury khromatina.

The correlation between the rates of nuclear polypeptide synthesis (NPS) and matrix protein synthesis in rat liver cells was investigated. It was shown that NPS is activated under conditions of protein synthesis inhibition in the cytoplasm. Model experiments revealed that the NPS and ADP ribosylation systems compete for chromatin structure: ADP ribosylation induces condensation, while NPS--decondensation of chromatin.