Heat-induced activation of reducing properties of of sea water

It was shown using the Ellman's reagent that chloride and bicarbonate anions are the heat-induced reducing agents of sea-water, and their combined action is more than additive. Sulfate anions do not exhibit these properties. The influence of sea-water anions on the heat-induced production of hydrogen peroxide was studied by enhanced chemiluminescence in a peroxidase-luminol-p-iodophenol system. In NaCl and NaHCO3 solutions, at concentration and pH values equal to those of sea-water, the production of H2O2 upon heating increased, as compared with water, whereas sulfate anions depressed its formation. By using coumarin-3-carboxylic acid as a fluorescent detector of OH radicals, a substantial increase in the production of radicals in the presence of chloride and bicarbonate anions upon heating was shown. The effect is due to the electron donor properties of these anions, which lead to the decomposition of H2O2 with the formation of OH radicals. The results obtained were considered from the viewpoint of the equivalence of heat and electromagnetic radiation of an absolutely black body. It is supposed that the high-energy quanta of its spectrum lead to the dissociation of anions with the formation of a hydrated electron and radicals. Then a recombination of radicals with the formation of various molecular products takes place.     

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 × 10^–2 µmol J^–1 for 8-oxoguanine production in DNA upon γ-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°C gave a value of k₃₇ = 4.7 × 10^–10 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.     

Level of hydrogen peroxide in electrochemically activated solutions and study of its effect on growth of Escherichia coli

The formation of hydrogen peroxide in catholytes and anolytes of electrochemically activated solutions: bidistilled water and solutions of sodium chloride and nutrition medium M9 was studied. The concentration of hydrogen peroxide was determined by the method of enhanced chemiluminescence in a system peroxidase-luminol-p-iodophenol. It was shown that the concentration of hydrogen peroxide depends on the ionic content of the solution and varies from a few fractions of a micromole in catholytes of bidistilled water and sodium chloride solutions (10(-5) divided by 10(-2) M) to 20-25 microM in catholytes of medium M9. The concentration of H2O2 in anolytes of various solutions was 15-20 times lower than in the corresponding catholytes and was equal to a few nanomoles in bidistilled water and a few micromoles in medium M9. The biological activity of the catholyte of medium M9 was determined from changes in the growth of E. coli cells. It was found that this catholyte stimulates the cell growth. The stimulating effect was 20-25% and did not change after the decomposition of hydrogen peroxide in the catholyte by catalase. The addition of H2O2 at the corresponding concentration to the inactivated nutrient medium produced no stimulating effect. These data suggest that hydrogen peroxide formed in the catholyte of nutrient medium M9 does not affect its biological activity.     

Study of formation and elimination of 8-oxoguanine in DNA of the liver and brain of mice after gamma-irradiation

8-oxoguanine (8-OG) is a biological marker of oxidative damage to DNA by reactive oxygen species. With the help of monoclonal antibodies to 8-OG, by the method of chemiluminescence enzyme immunoassay essential distinctions were found in formation and elimination of this damage to DNA in a liver and brain of mice exposed to 20 Gy of gamma-radiation. A spontaneous level of 8-OG in DNA of brain is much higher than in DNA of liver. Registered immediately after the exposure, the induction of 8-OG in DNA was approximately 3 times greater in brain than in liver. Elimination of 8-OG from DNA of both tissues proceeds more actively only during 30 min after the exposure, and the significant percentage (26-30%) of these damage is kept in DNA of the tissues for 3 hours after the exposure. The levels of lipidsoluble antioxidants, determined by the reaction with a stable free radical, in tissues of mice do not correlate with a quantity of 8-OG, arising in DNA spontaneously and under irradiation.     

Chemiluminescence enzyme immunoassay of 8-oxoguanine in DNA.

A test system has been developed to determine 8-oxoguanine in DNA, the most important biomarker of damage to DNA bases by reactive oxygen species. The system is based on a chemiluminescence enzyme immunoassay with the use of monoclonal antibodies (mcAB) against 8-oxoguanine. The test involves several stages: 1) immobilization of DNA on nitrocellulose membrane filters using an efficient technique with preliminary formation of a complex with protamine sulfate; 2) formation of antigen--antibody complexes (mcAB with 8-oxoguanine in DNA) with secondary antibodies and with a peroxidase--antiperoxidase complex (PAP method); 3) detection of increased chemiluminescence in a solution of hydrogen peroxide, luminol, and p-iodophenol. The increased chemiluminescence is determined with a conventional liquid scintillation counter for measuring beta-radioactivity. The system was tested by determining 8-oxoguanine formation in DNA upon gamma-irradiation and upon photosensitized oxidation of guanine under visible light in the presence of methylene blue. A linear dose dependence of 8-oxoguanine formation in DNA was shown for gamma-irradiation. The radiation-chemical yield of 8-oxoguanine (G = 0.57 molecule per 100 eV) is convenient to use for calibration of the amount of 8-oxoguanine formed under other conditions. The sensitivity of the method permits the detection of several femtomoles of 8-oxoguanine in a 40 microg sample of DNA.