Glucose oxidase-produced H 2O 2 induces Ca 2+-dependent DNA damage in human peripheral blood lymphocytes

Abstract

DNA of lymphocytes from human peripheral blood was analyzed by using the single cell gel electrophoresis technique (comet assay). The cells were used either as received from the donors or after treatment with various concentrations of the H 2O 2-generating enzyme glucose oxidase, in order to achieve a continuous flow of H 2O 2. The formation of single strand breaks (SSB) was dose-related but the time course of the induction of SSB by relatively low concentrations of glucose oxidase was of a biphasic mode with a fast increase 2 to 5 min after the addition of glucose oxidase followed by a gradual decrease toward the original base level during the next 35 to 60 min. This response of the cells appears to be based on the activation of already existing defense system(s) because it was shown that H 2O 2 is continuously released during the reaction time and the inhibition of protein synthesis does not affect the observed pattern. Supplementation of the growth medium with various antioxidants resulted in substantial protection only when the agents were taken up by the cells. The presence of the intracellular calcium chelator BAPTA protected the cells from H 2O 2-induced DNA damage in a dose-dependent manner. Only at the higher rate of H 2O 2-generation considerable DNA damage was observed in the presence of BAPTA.These results suggest that H 2O 2, at low concentrations induces DNA damage through intracellular Ca 2+-mediated processes, which lead to DNA strand breaks possibly by endonuclease activation.