It is demonstrated that hydroxyl radicals and hydrogen peroxide are formed under the action of uranyl ions in aqueous solutions containing no reducing agents. In the presence of uranyl ions, formation of 8-oxoguanine in DNA and long-lived protein radicals are observed in vitro. It is shown that the pro-oxidant properties of uranyl at micromolar concentrations mostly result from the physico-chemical nature of the compound rather than its radioactive decay. Uranyl ions lead to damage in DNA and proteins causing death of HEp-2 cells by necrotic pathway. It is revealed that the uranyl ions enhance radiation-induced oxidative stress and significantly increase a death rate of mice exposed to sublethal doses of X-rays.

Formation of long-lived radicals in solutions of casein and its hydrolysate with an equimolar mixture of amino acids was compared by measuring the X-ray-induced chemiluminescence. It was shown that free amino acids constituting the protein produce long-lived radicals. It was demonstrated that some amino acids (Leu, Ile, Val, Ser, Trp, Met, Pro, Arg, Gly, Phe) emit light of visible spectrum over a long period of time after irradiation, which indicates generation of long-lived radicals of these amino acids. The half-life times of these radicals are several hours. Dissolution of irradiated dry amino acids capable of luminescing over a long time causes formation of hydrogen peroxide in the aqueous medium.

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.

The content of 8-oxoguanine, a biomarker of DNA damage by the action of reactive oxygen species, in native and denatured DNA upon heating at 37 degrees C was studied by the enzyme-linked immunosorbent assay using monoclonal antibodies against 8-oxoguanine,. It was found that the content of 8-oxoguanine changes with time in a complicated multiphase manner, the maximum changes being as great as twofold. The production of hydrogen peroxide in water and 1 mM PBS, pH 6.8, at 37 degrees C over a period of 50 h was determined by the method of enhanced chemiluminescence in a peroxide-luminol-p-iodophenol system, The generation of hydrogen peroxide also changed in a complicated multiphase manner. After heating the DNA at 80 degrees C for 24 h, guanine oxidation products were excised by 8-oxoguanine-DNA-glycosylase. The products were separated and analyzed by liquid column chromatography on Sephadex LH-20. and Toyopearl HW-40 gel. The products were identified from UV adsorption spectra. The results indicated the generation of reactive oxygen. species at 37 degrees C, which leads both to the generation of 8-oxoguanine in DNA and its elimination as a result of its further oxidation. The oxidation of 8-oxoguanine was accompanied by the formation of a number of unstable products of further oxidation of 8-oxoguanine. Among these products, aminoimidazolone, spiroiminodigidantoin, and diiminoimidazole were identified from UV spectra. The appearance of the products of further oxidation of 8-oxoguanine explains the origin of G : C -> C : G transversions by the action of reactive oxygen species.

8-Oxoguanine (8-OG) is an important biomarker of oxidative DNA damage induced by reactive oxygen species (ROS). By using ELISA with monoclonal antibodies against 8-OG, the formation of 8-OG in DNA by the action of uranyl ions, gamma-irradiation, and heating at 37 degrees C and their combined action was investigated in view of environmental pollution by uranium oxides as a result of the use of armor piercing shells with depleted uranium. The content of 8-OG in DNA induced by the action of gamma-irradiation, 5 microM uranyl ions and heating changes with time in a complicated manner. These results suggest that, by the action of uranyl ions, an additional generation of ROS occurs, which leads both to the formation of 8-OG in DNA and its further oxidation. Uranyl ions at a conceptration of 5 microM increase the thermal deamination of cytosine in DNA several times but do not influence DNA thermal depurination. It is shown that uranyl ions essentially increase the production of hydrogen peroxide and hydroxyl radicals by the action of heat on water. The results indicate a high chemical genotoxicity of uranyl ions and their enhancing effect on DNA base damage by the action of heat and gamma-irradiation.

The content of 8-oxoguanine, a biomarker of DNA damage by the action of reactive oxygen species, in native and denatured DNA upon heating at 37 degrees C was studied by the enzyme-linked immunosorbent assay using monoclonal antibodies against 8-oxoguanine. It was found that the content of 8-oxoguanine changes with time in a complicated multiphase manner, the maximum changes being as great as twofold. The production of hydrogen peroxide in water and 1 mM PBS, pH 6.8, at 37 degrees C over a period of 50 h was determined by the method of enhanced chemiluminescence in a peroxide-luminol-p-iodophenol system. The generation of hydrogen peroxide also changed in a complicated multiphase manner. After heating the DNA at 80 degrees C for 24 h, guanine oxidation products were excised by 8-oxoguanine-DNA-glycosylase. The products were separated and analyzed by liquid column chromatography on Sephadex LH-20 and Toyopearl HW-40 gel. The products were identified from UV adsorption spectra. The results indicated the generation of reactive oxygen species at 37 degrees C, which leads both to the generation of 8-oxoguanine in DNA and its elimination as a result of its further oxidation. The oxidation of 8-oxoguanine was accompanied by the formation of a number of unstable products of further oxidation of 8-oxoguanine. Among these products, aminoimidazolone, spiroiminodigidantoin, and diiminoimidazole were identified from UV spectra. The appearance of the products of further oxidation of 8-oxoguanine explains the origin of G : C --> C : G transversions by the action of reactive oxygen species.