DNA damage and apoptosis in hydrogen peroxide-exposed Jurkat cells: bolus addition versus continuous generation of H(2)O(2).
Aspects of the molecular mechanism(s) of hydrogen peroxide-induced DNA damage and cell death were studied in the present investigation. Jurkat T-cells in culture were exposed either to low rates of continuously generated H(2)O(2) by the action of glucose oxidase or to a bolus addition of the same agent. In the first case, steady state conditions were prevailing, while in the latter, H(2)O(2) was removed by the cellular defense systems following first order kinetics. By using single-cell gel electrophoresis (also called comet assay), an initial increase in the formation of DNA single-strand breaks was observed in cells exposed to a bolus of 150 microM H(2)O(2). As the H(2)O(2) was exhausted, a gradual decrease in DNA damage was apparent, indicating the existence of an effective repair of single-strand breaks. Addition of 10 ng glucose oxidase in 100 microl growth medium (containing 1.5 x 10(5) cells) generated 2.0 +/- 0.2 microM H(2)O(2) per min. This treatment induced an increase in the level of single-strand breaks reaching the upper limit of detection by the methodology used and continued to be high for the following 6 h. However, when a variety of markers for apoptotic cell death (DNA cell content, DNA laddering, activation of caspases, PARP cleavage) were examined, only bolus additions of H(2)O(2) were able to induce apoptosis, while the continuous presence of this agent inhibited the execution of the apoptotic process no matter whether the inducer was H(2)O(2) itself or an anti-Fas antibody. These observations stress that, apart from the apparent genotoxic and proapoptotic effects of H(2)O(2), it can also exert antiapoptotic actions when present, even at low concentrations, during the execution of apoptosis.
Barbouti
Alexandra
A
Laboratory of Biological Chemistry, University of Ioannina Medical School, Ioannina, Greece.
Doulias
Paschalis-Thomas
PT
Nousis
Lambros
L
Tenopoulou
Margarita
M
Galaris
Dimitrios
D
D017209:Apoptosis; D015153:Blotting, Western; D020169:Caspases / Q000378:metabolism; D002470:Cell Survival; D004396:Coloring Agents / Q000494:pharmacology; D020552:Comet Assay; D004249:DNA Damage; D053938:DNA Fragmentation; D004789:Enzyme Activation; D005434:Flow Cytometry; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D019169:Jurkat Cells; D011065:Poly(ADP-ribose) Polymerases; D013778:Tetrazolium Salts / Q000494:pharmacology; D013844:Thiazoles / Q000494:pharmacology; D013997:Time Factors
D004396:Coloring Agents; D013778:Tetrazolium Salts; D013844:Thiazoles; D006861:Hydrogen Peroxide; D011065:Poly(ADP-ribose) Polymerases; D020169:Caspases; C022616:thiazolyl blue