Laser Ablation-Generated Crystalline Selenium Nanoparticles Prevent Damage of DNA and Proteins Induced by Reactive Oxygen Species and Protect Mice against Injuries Caused by Radiation-Induced Oxidative Stress.

With the help of laser ablation, a technology for obtaining nanosized crystalline selenium particles (SeNPs) has been created. The SeNPs do not exhibit significant toxic properties, in contrast to molecular selenium compounds. The administration of SeNPs can significantly increase the viabilities of SH-SY5Y and PCMF cells after radiation exposure. The introduction of such nanoparticles into the animal body protects proteins and DNA from radiation-induced damage. The number of chromosomal breaks and oxidized proteins decreases in irradiated mice treated with SeNPs. Using hematological tests, it was found that a decrease in radiation-induced leukopenia and thrombocytopenia is observed when selenium nanoparticles are injected into mice before exposure to ionizing radiation. The administration of SeNPs to animals 5 h before radiation exposure in sublethal and lethal doses significantly increases their survival rate. The modification dose factor for animal survival was 1.2. It has been shown that the introduction of selenium nanoparticles significantly normalizes gene expression in the cells of the red bone marrow of mice after exposure to ionizing radiation. Thus, it has been demonstrated that SeNPs are a new gene-protective and radioprotective agent that can significantly reduce the harmful effects of ionizing radiation.

Metformin mitigates radiation toxicity exerting antioxidant and genoprotective properties.

The antidiabetic drug metformin (MF) exhibits redox-modulating effects in various pathologies associated with oxidative stress and mitigates ionizing radiation-induced toxicity, but the underlying mechanisms remain to be elucidated. Thus, we studied some radiomitigatory effects of MF and explored the possible mechanisms behind them. Highly sensitive luminescence methods and non-competitive enzyme-linked immunosorbent assay (ELISA) were used in in vitro studies, and in vivo the damage to bone marrow cells and its repair were assessed by the micronucleus test. In a solution, MF at concentrations exceeding 0.1 µM effectively intercepts •OH upon X-ray-irradiation, but does not react directly with H2O2. MF accelerates the decomposition of H2O2 catalyzed by copper ions. MF does not affect the radiation-induced formation of H2O2 in the solution of bovine gamma-globulin (BGG), but has a modulating effect on the generation of H2O2 in the solution of bovine serum albumin (BSA). MF at 0.05-1 mM decreases the radiation-induced formation of 8-oxoguanine in a DNA solution depending on the concentration of MF with a maximum at 0.25 mM. MF at doses of 3 mg/kg body weight (bw) and 30 mg/kg bw administered to mice after irradiation, but not before irradiation, reduces the frequency of micronucleus formation in polychromatophilic erythrocytes of mouse red bone marrow. Our work has shown that the radiomitigatory properties of MF are mediated by antioxidant mechanisms of action, possibly including its ability to chelate polyvalent metal ions.

Bacteriostatic and Cytotoxic Properties of Composite Material Based on ZnO Nanoparticles in PLGA Obtained by Low Temperature Method.

A low-temperature technology was developed for producing a nanocomposite based on poly (lactic-co-glycolic acid) and zinc oxide nanoparticles (ZnO-NPs), synthesized by laser ablation. Nanocomposites were created containing 0.001, 0.01, and 0.1% of zinc oxide nanoparticles with rod-like morphology and a size of 40-70 nm. The surface of the films from the obtained nanomaterial was uniform, without significant defects. Clustering of ZnO-NPs in the PLGA matrix was noted, which increased with an increase in the concentration of the dopant in the polymer. The resulting nanomaterial was capable of generating reactive oxygen species (ROS), such as hydrogen peroxide and hydroxyl radicals. The rate of ROS generation increased with an increase in the concentration of the dopant. It was shown that the synthesized nanocomposite promotes the formation of long-lived reactive protein species, and is also the reason for the appearance of a key biomarker of oxidative stress, 8-oxoguanine, in DNA. The intensity of the process increased with an increase in the concentration of nanoparticles in the matrix. It was found that the nanocomposite exhibits significant bacteriostatic properties, the severity of which depends on the concentration of nanoparticles. In particular, on the surface of the PLGA-ZnO-NPs composite film containing 0.001% nanoparticles, the number of bacterial cells was 50% lower than that of pure PLGA. The surface of the composite is non-toxic to eukaryotic cells and does not interfere with their adhesion, growth, and division. Due to its low cytotoxicity and bacteriostatic properties, this nanocomposite can be used as coatings for packaging in the food industry, additives for textiles, and also as a material for biomedicine.

Unmodified hydrated С60 fullerene molecules exhibit antioxidant properties, prevent damage to DNA and proteins induced by reactive oxygen species and protect mice against injuries caused by radiation-induced oxidative stress.

Unmodified hydrated С60 fullerene molecules (C60UHFM) were shown to reduce the formation ROS in water and 8-oxoguanine in DNA upon ionizing radiation impact. C60UHFM efficiently eliminate long-lived protein radicals arising after irradiation. In irradiated mice C60UHFM reduce the rate of single/double-strand DNA breaks and amount of chromosomal breaks. The radioprotective activity of C60UHFM was estimated by the survival rate of animals; the dose modification factor for animal survival was 1.3. Hematological tests showed that C60UHFM injection in mice prior to irradiation results in a decrement of irradiation-induced leucopenia and thrombocytopenia. Histological analysis testified that C60UHFM provide significant protection of small intestine tissues in mice against irradiation-induced damage. The obtained data assume that the radioprotective properties of C60UHFM are determined by their antioxidant, antiradical and DNA-protective qualities. Thus, it was demonstrated that C60UHFM are a novel antioxidant and radioprotective agent capable of substantial reduction of the harmful effects of ionizing radiation.

Formation of long-lived reactive species of blood serum proteins induced by low-intensity irradiation of helium-neon laser and their involvement in the generation of reactive oxygen species.

It was demonstrated that low-intensity radiation of helium-neon (He-Ne) laser at 632.8nm, which leads to the transition of oxygen to a singlet state, causes the formation of reactive oxygen species (ROS) - hydrogen peroxide, hydroxyl and superoxide (hydroperoxide) radicals - in aqueous solutions. The oxygen effect - dependence of hydrogen peroxide formation on the concentration of molecular oxygen - was shown, and the participation of singlet oxygen, hydroxyl radicals and superoxide (hydroperoxide) radicals in this process was testified. Laser radiation-induced ROS in solutions of blood serum proteins, bovine serum albumin and bovine gamma-globulin, cause the formation of long-lived reactive protein species (LRPS) with a half-life of about 4h. The generation of LRPS caused by laser irradiation results in prolonged several-hour generation of ROS - hydrogen peroxide, hydroxyl and superoxide radicals. As affected by LRPS, coupled radical reactions lead to conversion of dissolved molecular oxygen to hydrogen peroxide. Irradiation with light sources away from the oxygen absorption band is not attended by formation of ROS and LRPS. A consideration is provided for the possible molecular mechanisms of ROS formation under the influence of He-Ne laser irradiation, the role of proteins in their generation and the biological significance of these processes.

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.

Self-oscillating Water Chemiluminescence Modes and Reactive Oxygen Species Generation Induced by Laser Irradiation; Effect of the Exclusion Zone Created by Nafion.

Samples of water inside and outside an exclusion zone (EZ), created by Nafion swollen in water, were irradiated at the wavelength l = 1264 nm, which stimulates the electronic transition of dissolved oxygen from the triplet state to the excited singlet state. This irradiation induces, after a long latent period, chemiluminescence self-oscillations in the visible and near UV spectral range, which last many hours. It occurs that this effect is EZ-specific: the chemiluminescence intensity is twice lower than that from the bulk water, while the latent period is longer for the EZ. Laser irradiation causes accumulation of H2O2, which is also EZ-specific: its concentration inside the EZ is less than that in the bulk water. These phenomena can be interpreted in terms of a model of decreasing O2 content in the EZ due to increased chemical activity of bisulfite anions (HSO3-), arisen as the result of dissociation of terminal sulfonate groups of the Nafion. The wavelet transform analysis of the chemiluminescence intensity from the EZ and the bulk water gives, that self-oscillations regimes occurring in the liquid after the latent period are the determinate processes. It occurred that the chemiluminescence dynamics in case of EZ is characterized by a single-frequency self-oscillating regime, whereas in case of the bulk water, the self-oscillation spectrum consists of three spectral bands.

Heat-induced formation of nitrogen oxides in water.

It was found by the fluorimetric method using 2,3-diaminonaphthalene that moderate heating of water (60-80°C, for up to 4 h) leads to the fixation of atmospheric nitrogen with the formation of nitrite. The kinetic parameters of this process were determined. The energy of activation of [Formula: see text]formation was estimated to be 139 kJ/mol. It was found that the amount of nitrite formed depends on the concentration of dissolved oxygen and nitrogen. It was shown by two independent methods (Griess reagent/VCl3 and 2,3-diaminonaphthalene/nitrate reductase) that heating of water (80°C, 1 h) results in the formation of nitrate; with the use of the fluorescent probe dihydrorhodamine 123, the generation of nitrogen dioxide (peroxynitrite) was revealed. Nitrite, nitrate, and nitrogen dioxide are formed in water upon heating in approximately equal amounts. A scheme of reactions proceeding with bidistilled water by the action of heat with the formation of nitrogen oxides is proposed.

Prolongation of oxidative stress by long-lived reactive protein species induced by X-ray radiation and their genotoxic action.

The formation of long-lived reactive protein species of bovine serum albumin (BSA), ovalbumin, casein and casein hydrolyzate with a half-life of 3-5 hours was shown using chemiluminescence induced by X-ray radiation. It was found that long-lived reactive protein species are capable of generating reactive oxygen species (ROS) (H2O2, OH(•), HO2(•)¹O2) in the aquatic environment over a long period of time in vitro. The interaction of X-ray-irradiated BSA with DNA in vitro led to the formation of 8-oxoguanine (8-oxo-7,8-dihydroguanine), a biomarker of oxidative damage to DNA. Some natural antioxidants are effective scavengers of ROS (inosine, tryptophan, methionine and ascorbate). They protect DNA from the action of long-lived reactive protein species leading to ROS generation and the formation of 8-oxoguanine. The intravenous injection of X-ray radiation-induced, long-lived reactive protein species to rats, as well as the peroral and intraperitoneal administration of these products to mice, gave rise to cytogenetic injuries in the cells of their red bone marrow through the formation of micronuclei in polychromatophilic erythrocytes. The administration of the same natural antioxidants used for in vitro experiments soon after irradiation made it possible to effectively eliminate the genotoxic action of oxidative stress caused by radiation-induced, long-lived reactive protein species. Our data represent clear evidence that the oxidative damage to proteins induced by X-rays is directly involved in the induction of a response to DNA damage in rodents.

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.

Peculiarities of the antioxidant and radioprotective effects of hydrated C60 fullerene nanostuctures in vitro and in vivo.

Aqueous solutions of highly stable supramolecular donor-acceptor complexes of chemically nonmodified pristine C(60) fullerene molecules with H(2)O molecules (hydrated C(60) fullerene-C(60)HyFn) and their labile nano-sized clusters were examined for their antioxidant effects on removal of hydroxyl radicals (.OH) and protecting DNA against oxidative damage induced by ionizing radiation in vitro. The suppressing influence of C(60)HyFn on the formation of OH-radicals in water exposed to X-rays at doses of 1-7 Gy was assessed by determination of oxidation levels of coumarin-3-carboxylic acid. C(60)HyFn demonstrates apparent antiradical activity in vitro in the range of concentrations of 10(-11)-10(-6) M. Paradoxically, the .OH-removing efficacy of C(60)HyFn was in reverse correlation with fullerene concentration. It was hypothesized that the antiradical action of C(60)HyFn in water medium generally is due to a "nonstoichiometric" mechanism, supposedly to a hydrated free radical recombination (self-neutralization), which is catalyzed by specific water structures ordered by C(60)HyFn. With the use of 8-oxoguanine as a marker of oxidative damage to DNA, it has been demonstrated that C(60)HyFn in concentrations of 10(-7)-10(-6) M protects nucleic acids against radical-induced damage. The second part of the present study was aimed to evaluate the overall radioprotective efficacy of C(60)HyFn in doses of 0.1 or 1 mg/kg b.w. injected intraperitoneally to mice either 1 h before or 15 min after lethal dose exposure of the X-ray (7 Gy) irradiation. Survival rate of the mice was observed at 30 day intervals after irradiation, while the weight gains of experimental animals were monitored as well. The most significant protective effect was demonstrated when 1 mg/kg dosage of C(60)HyFn was administered before irradiation. The outcome of the substance testing is 15% survival rate of irradiated animals at 30 days of observation, and prevention of noticeable weight loss characteristic for radiation impact, versus unprotected control animals. In conclusion, results of the study obviate that the apparent protective action of C(60)HyFn in vivo is determined by its considerable ability to decrease X-ray-generated reactive oxygen species. Based on the results and that neat C(60) is nontoxic, actually in the hydrated form, without side effects and with sufficient radioprotective effects in low doses, C(60)HyFn may be considered as a novel antioxidant agent, which substantially diminishes the harmful effects of ionizing radiation.

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 ( approximately 30 microg g(-1), i.e., approximately 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.

Guanosine and inosine display antioxidant activity, protect DNA in vitro from oxidative damage induced by reactive oxygen species, and serve as radioprotectors in mice.

The effect of ribonucleosides on 8-oxoguanine formation in salmon sperm DNA dissolved in 1 mM phosphate buffer, pH 6.8, upon exposure to gamma 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 degrees 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 gamma-radiation-induced death.

Heat-induced formation of reactive oxygen species and 8-oxoguanine, a biomarker of damage to DNA.

Heat-induced formation of 8-oxoguanine was demonstrated in DNA solutions in 10(-3) M phosphate buffer, pH 6.8, by enzyme-linked immunosorbent assays using monoclonal antibodies against 8-oxoguanine. A radiation-chemical yield of 3.7 x 10(-2) micromol x J(-1) for 8-oxoguanine production in DNA upon gamma-irradiation was used as an adequate standard for quantitation of 8-oxoguanine in whole DNA. The initial yield of heat-induced 8-oxoguanine exhibits first order kinetics. The rate constants for 8-oxoguanine formation were determined at elevated temperatures; the activation energy was found to be 27 +/- 2 kcal/mol. Extrapolation to 37 degrees C gave a value of k37 = 4.7 x 10(-10) x s(-1). Heat-induced 8-oxoguanine formation and depurination of guanine and adenine show similarities of the processes, which implies that heat-mediated generation of reactive oxygen species (ROS) should occur. Heat-induced production of H2O2 in phosphate buffer was shown. The sequence of reactions of thermally mediated ROS formation have been established: activation of dissolved oxygen to the singlet state, generation of superoxide radicals and their dismutation to H2O2. Gas saturation (O2, N2 and Ar), D2O, scavengers of 1O2, O2-* and OH* radicals and metal chelators influenced heat-induced 8-oxoguanine formation as they affected thermal ROS generation. These findings imply that heat acts via ROS attack leading to oxidative damage to DNA.