S. V. Gudkov

Guanosine and Inosine Display Antioxidant Activity, Protect DNA In Vitro from Oxidative Damage Induced by Reactive Oxygen Species, and Serve as Radioprotectors in Mice

Abstract Gudkov, S. V., Shtarkman, I. N., Smirnova, V. S., Chernikov, A. V. and Bruskov, V. I. Guanosine and Inosine Display Antioxidant Activity, Protect DNA In Vitro from Oxidative Damage Induced by Reactive Oxygen Species, and Serve as Radioprotectors in Mice. Radiat. Res. 165, 538–545 (2006). The effect of ribonucleosides on 8-oxoguanine formation in salmon sperm DNA dissolved in 1 mM phosphate buffer, pH 6.8, upon exposure to γ rays was examined by ELISA using monoclonal antibodies against 8-oxoguanine. Nucleosides (1 mM) decreased the radiation-induced yield of 8-oxoguanine in the order Guo > Ino > Ado > Thd > Urd > Cyd. Guanosine and inosine considerably reduced deamination of cytosine in the DNA solutions upon heating for 24 h at 80°C. The action of nucleosides on the heat-induced generation of reactive oxygen species in the phosphate buffer was studied. The concentration of hydrogen peroxide was measured by enhanced chemiluminescence in a peroxidase–luminol–p-iodophenol system; the hydroxyl radical formation was measured fluorometrically by the use of coumarin-3-carboxylic acid. Guanosine and inosine considerably decreased the heat-induced production of both hydrogen peroxide and OH radicals. Guanosine and inosine increased survival of mice after a lethal dose of radiation. They especially enhanced the survival of animals when were administered shortly after irradiation. The results indicate that guanosine and inosine, natural antioxidants, prevent oxidative damage to DNA, decrease the generation of ROS, and protect mice against γ-radiation-induced death.

Oxygen-Dependent Auto-Oscillations of Water Luminescence Triggered by the 1264 nm Radiation

A 5-min exposure of air-saturated bidistilled water to low-intensity laser infrared radiation at the wavelength of the electronic transition of dissolved oxygen to the singlet state ((3)∑(g)(-)→ (1)Δ(g)) induces, after a long latent period, auto-oscillations of water luminescence in the blue-green region, which last many hours. Laser irradiation causes the accumulation of hydrogen peroxide, which depends on the concentration of dissolved oxygen. The auto-oscillations do not arise if water is irradiated beyond the oxygen absorption band and if the oxygen is removed from water. The wavelet transform analysis of luminescence records indicates that there are two characteristic periods of pulsations of about 300 and 1150 s. The results obtained suggest that auto-oscillations are triggered by photoinduced singlet oxygen (1)Δ(g), and this phenomenon is closely related to formation of hydrogen peroxide.

Protection of mice against X-ray injuries by the post-irradiation administration of guanosine and inosine

Purpose: To examine the radioprotective action of guanosine (Guo) and inosine (Ino) administered to mice after irradiation with X-rays. Materials and methods: Survival of mice exposed to lethal and sublethal doses of X-rays was studied. Peripheral blood cells were counted using a light microscope. The damage to bone marrow cells was assessed by micronucleus (MN) test. Damage and repair of DNA in blood leukocytes were estimated using the comet assay. Results: Mice injected intraperitoneally (i.p.) with Guo or Ino (∼30 μg g−1, i.e., ∼0.6 mg per 20-g mouse) 15 min after acute whole-body irradiation with 7 Gy recovered from X-ray injury. On the 30th day after irradiation, 50 and 40% of mice injected with Guo and Ino, respectively, remained alive. The dose reduction factor (DRF) was 1.23 for Guo and 1.15 for Ino. The protective effect gradually decreased as the time interval between the irradiation and injection was increased to 3, 5, 8 h. Guo and Ino facilitated the restoration of peripheral blood cell counts. These compounds protected bone marrow cells from damage and normalized erythropoiesis. Guo and Ino contributed to a more rapid and complete repair of DNA in mouse leukocytes irradiated both in vitro and in vivo. Conclusion: Guo and Ino introduced shortly after irradiation reduce leukopenia and thrombocytopenia and offer promise as therapeutic agents for treatment of radiation injuries.

Targeted Radionuclide Therapy of Human Tumors

Targeted radionuclide therapy is one of the most intensively developing directions of nuclear medicine. Unlike conventional external beam therapy, the targeted radionuclide therapy causes less collateral damage to normal tissues and allows targeted drug delivery to a clinically diagnosed neoplastic malformations, as well as metastasized cells and cellular clusters, thus providing systemic therapy of cancer. The methods of targeted radionuclide therapy are based on the use of molecular carriers of radionuclides with high affinity to antigens on the surface of tumor cells. The potential of targeted radionuclide therapy has markedly grown nowadays due to the expanded knowledge base in cancer biology, bioengineering, and radiochemistry. In this review, progress in the radionuclide therapy of hematological malignancies and approaches for treatment of solid tumors is addressed.

Generation of reactive oxygen species in water under exposure to visible or infrared irradiation at absorption bands of molecular oxygen

It is found that in bidistilled water saturated with oxygen, hydrogen peroxide and hydroxyl radicals are formed under the influence of visible and infrared radiation in the absorption bands of molecular oxygen. Formation of reactive oxygen species (ROS) occurs under the influence of both solar and artificial light sources, including the coherent laser irradiation. The oxygen effect, i.e. the impact of dissolved oxygen concentration on production of hydrogen peroxide induced by light, is detected. It is shown that the visible and infrared radiation in the absorption bands of molecular oxygen leads to the formation of 8-oxoguanine in DNA in vitro. Physicochemical mechanisms of ROS formation in water when exposed to visible and infrared light are studied, and the involvement of singlet oxygen and superoxide anion radicals in this process is shown.

Guanosine and Inosine (Riboxin) Eliminate the Long-Lived Protein Radicals Induced X-ray Radiation

A considerable part of damages of biopolymers incells, occurring under exposure to ionizing radiation,are due to short-lived radicals produced as a result ofwater radiolysis [1]. EPR spectroscopic studies showedthat exposure to ionizing radiation leads to generationof long-lived protein radicals in protein solutions andcells [2–11]. The half-life of these radicals reaches 20[2] and more hours [11]. The occurrence of long-livedradicals was demonstrated in model systems for someproteins, such as egg albumin [3, 5, 6], bovine serumalbumin [7, 12], human serum albumin [9], lysozyme[12], immunoglobulin G [9], and histone H1 [10].Long-lived radicals are formed under exposure togamma- [3–6, 10, 11], X-ray [2], and ultraviolet radia-tion [8], as well as in the presence of peroxynitrite [9]and the products of hydrogen peroxide decompositionby immobilized peroxidase [7]. The formation of suchradicals was detected in human blood plasma proteins[9]; human [2] and hamster [5] embryonic cells; Droso-phila germ cells; and in root, seed, and leaf cells of gar-den radish and Arabidopsis [4].It was established that the long-lived protein radi-cals may be the sources of generation of reactive oxy-gen species and long-term oxidative stress in biologicalsystems [7] and function as messengers transmittingoxidative damages to other molecules, including DNA[10]. An exposure of histone H1 to gamma-radiationyields radicals that are involved in the formation ofDNA–protein crosslinks and oxidative damage of basesin DNA, producing the mutagenic product 8-oxogua-nine [10]. It was shown that the long-lived protein rad-icals induce mutations and lead to cell transformation[2, 3]. However, normally these radicals are producedin small quantities in living animal and plant cells [4].The long-lived protein radicals can be immediatelyrecorded using ESR spectroscopy; however, a low sen-sitivity of this method makes it possible to detect theformation of such radicals only at very high radiationdoses (1–5 kGy) [3, 5, 6, 10, 11]. Another effective andsensitive method to detect free-radical reactions ischemiluminescence, when interaction of radicals yieldsenergy emitted in the form of light quanta [1]. In thiswork, we detected and studied long-lived protein radi-cals by measuring the intrinsic chemiluminescence ofprotein solutions induced by X-ray radiation using ahigh-sensitivity Biotoks-7A chemiluminometer (Rus-sia). Measurements were performed in 20-ml plasticpolypropylene vials for a liquid scintillation counter(Beckman, United States). The use of larger volumesfor measuring chemiluminescence compared to theconventionally used volumes (at most 0.1 ml) allowedus to significantly (more than 200 times) increase thesensitivity of this method and to detect the formation ofprotein radicals at doses of several Gy. To study chemi-luminescence, we used solutions of ovalbumin (Rea-Khim, Russia) prepared in 20 mM Tris-HCl buffer(pH 8.0). The samples were irradiated using a RUT-15therapeutic X-ray device (Russia) under the followingconditions: voltage, 200 kV; amperage, 20 mA; focusdistance, 37.5 cm. Samples containing unirradiatedprotein and protein-free irradiated samples were usedas controls. The dependence of chemiluminescenceintensity on ovalbumin concentration was bell-shaped;the maximum chemiluminescence intensity (irradiationdose, 7 Gy) was detected for 0.5% ovalbumin. This pro-tein concentration was used in further experiments.To rule out the effect of light quanta as a result ofrecombination of radical occurring upon decomposi-tion of hydrogen peroxide and other possible productsformed in the buffer, the chemiluminescence intensitywas measured 1 h after irradiation. During this time,samples were kept at room temperature in the dark.Because water radiolysis yields hydrogen peroxide,which has a long half-life, we studied the possibility ofinfluence of hydrogen peroxide on the chemilumines-cence yield in irradiated protein solution. We found that

Effect of amino acids on X-ray-induced hydrogen peroxide and hydroxyl radical formation in water and 8-oxoguanine in DNA

Generation of hydrogen peroxide and hydroxyl radicals in L-amino acid solutions in phosphate buffer, pH 7.4, under X-ray irradiation was determined by enhanced chemiluminescence in the luminol-p-iodophenol-peroxidase system and using the fluorescent probe coumarin-3-carboxylic acid, respectively. Amino acids are divided into three groups according to their effect on the hydrogen peroxide formation under irradiation: those decreasing yield of H2O2, having no effect, and increasing its yield. All studied amino acids at 1 mM concentration decrease the yield of hydroxyl radicals in solution under X-ray irradiation. However, the highest effect is observed in the order: Cys > His > Phe = Met = Trp > Tyr. At Cys, Tyr, and His concentrations close to physiological, the yield of hydroxyl radicals decreases significantly. Immunoenzyme analysis using monoclonal antibodies to 8-oxoguanine (8-oxo-7,8-dihydroguanine) was applied to study the effect of amino acids with the most pronounced antioxidant properties (Cys, Met, Tyr, Trp, Phe, His, Lys, Arg, Pro) on 8-oxoguanine formation in vitro under X-ray irradiation. It is shown that amino acids decrease the content of 8-oxoguanine in DNA. These amino acids within DNA-binding proteins may protect intracellular DNA against oxidative damage caused by formation of reactive oxygen species in conditions of moderate oxidative stress.

Formation of long-lived reactive species of blood serum proteins by the action of heat.

It has been previously established that heat induces the formation of reactive oxygen species (ROS) in aqueous solutions. In biological systems, ROS cause oxidative damage predominantly to proteins due to their abundance and sensitivity to oxidation. Proteins oxidized by the action of X-rays represent long-lived reactive species, which trigger the secondary generation of ROS (Bruskov et al. (2012) [25]). Here we studied the possibility of formation of long-lived species of the blood serum proteins bovine serum albumin and bovine gamma-globulin in air-saturated solutions under the action of heat. It is shown that heat induces the generation of long-lived protein species, which in turn generate ROS ((1)О2, (·)O2(-), (·)OН, and H2O2). The formation of the long-lived reactive species of BSA and BGG with a half-life of about 4h induced by moderate hyperthermia was revealed using the chemiluminescence of protein solutions. It was found that long-lived reactive species of BSA and BGG cause prolonged generation of H2O2. The results obtained suggest that H2O2 produced by proteins after heating represents a messenger in signaling pathways and produces therapeutic effects in living organisms.

Long-lived protein radicals induced by X-ray irradiation are the source of reactive oxygen species in aqueous medium.

), the mostreactive ROS [2]. In addition, the content of proteinsin cells (approximately 15%) is higher than the content of other biopolymers. It is believed that proteinoxidation by free radicals underlies cell aging andinduces a number of human diseases [3]. However, therole of ionizing radiationinduced oxidative damagesof proteins in the development of oxidative stress incells has been insufficiently studied.Protein damage by ionizing radiation in the presence of oxygen yields a number of oxidized products[4]. EPR spectroscopic studies showed that irradiationwith high doses of ionizing radiation (kGy) generateslonglived protein radicals (LLPRs) in cells and solutions of various proteins [5, 6] whose lifetime mayreach 20 h [6]. LLPRs were also recorded by measuring intrinsic chemiluminescence of proteins [7]. Thisapproach makes it possible to detect LLPR formationat irradiation doses from 1 to 10 Gy. The lifetime ofradicals of proteins (ovalbumin, bovine serum albumin(BSA), and casein) determined by this method at thespecified irradiation doses is approximately 3–5 h [7].Some studies demonstrated the possibility of transition of oxidative damages from LLPRs to DNA invitro to form the mutagenic product 8oxoguanine, amarker of DNA lesions induced by ROS [7–9]. It wasestablished that in vivo LLPRs induce mutations andlead to cell transformation [5, 6]. The most probablemechanism of LLPRinduced damage of other biological molecules, including DNA [7–9], may includeradicalinduced ROS production in aqueous solutionafter irradiation [10]. In this work, we studied the ability of longlived BSA radica ls induced by Xray irradiation to generate ROS (hydrogen peroxide andhydroxyl radicals) in aqueous solution and attemptedto determine possible molecular mechanism of thisprocesses.To measure the ability of LLPRs to Н

ANTIOXIDATIVE AND RADIATION MODULATING PROPERTIES OF GUANOSINE-5-MONOPHOSPHATE

Employing enhanced chemiluminescence in luminol-p-iodophenol peroxidase system and coumarine-3-carboxylic acid, it was shown that guanosine-5′-monophosphate (GMP) appreciably reduces formation of H2O2 and hydroxyl radicals induced by x-ray irradiation. Using immunoenzyme assay, we revealed that GMP lowered 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo) formation in DNA in vitro after irradiation. The results of survival test have shown that mice being injected intraperitoneally with GMP after irradiation with a dose of 7 Gy had better survival rate than the control mice. GMP reduced leucopoenia and thrombocytopenia in irradiated mice. Obtained results give premises that GMP may be promising therapeutic agent for treatment of radiation injuries.