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

Abstract

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.

Figure 1

Chromatographic separation of bases released from heat-treated DNA on a Toyopearl HW-40 column. DNA from chicken erythrocytes was dissolved at 355 µg/ml in 10^–3 M phosphate buffer, pH 6.8, and heated for 22 h at 90°C. Abscissa, time (min); marks on the axis correspond to 12 min intervals. Ordinate, optical density determined at 254 nm. The arrow indicates a 20-fold increase in recording sensitivity.

Figure 2

Arrhenius plots of –logk versus inverse temperature 1/T (k is the rate constant, s^–1; T is the temperature, K). 1, Guanine depurination in 10^–3 M phosphate buffer, pH 6.8; 2, 8-oxoguanine formation in 10^–3 M phosphate buffer, pH 6.8; 3, guanine depurination in 10^–3 M phosphate buffer, pH 6.8, with 0.14 M NaCl; 4, 8-oxoguanine formation in 10^–3 M phosphate buffer, pH 6.8, with 0.14 M NaCl.

Figure 3

Dose–yield relationship for the formation of 8-oxoguanine in γ-irradiated salmon sperm DNA obtained by competitive ELISA. Doses are 1, 2, 5, 10, 20 and 30 Gy. Background 8-oxoguanine level was subtracted from the observed data.

Figure 4

Kinetics of 8-oxoguanine formation in salmon sperm DNA on heating at different temperatures obtained by competitive ELISA: 1, 65°C; 2, 70°C; 3, 75°C; 4, 85°C. Background 8-oxoguanine level was subtracted from the observed data.

Figure 5

Standard calibration curve for quantitation of hydrogen peroxide in the nanomolar concentration range in 10^–3 M phosphate buffer, pH 6.8, obtained by competitive ELISA. Counts per minute (c.p.m.) versus nM H₂O₂.

Figure 6

Effects of SOD and catalase on heat-induced H₂O₂ formation in 10^–3 M phosphate buffer, pH 6.8. Samples of the phosphate buffer were heated at 75°C for 4 h and rapidly cooled to ambient temperature, then enzyme was added. The moment of enzyme addition corresponds to the zero point on the abscissa. 1, Phosphate buffer (control); 2, phosphate buffer with SOD; 3, phosphate buffer with catalase. Abscissa, incubation time at ambient temperature (26°C) (min). Ordinate, concentration of H₂O₂ (nM).