Glucose oxidase-produced H2O2 induces Ca2+-dependent DNA damage in human peripheral blood lymphocytes

DNA of lymphocytes from human peripheral blood was analyzed by using the si ngle cell gel electrophoresis technique (comet assay). The cells were used either as received from the donors or after treatment with various concentr ations of the H2O2-generating enzyme glucose oxidase, in order to achieve a continuous flow of H2O2. The formation of single strand breaks (SSB) was d ose-related but the time course of the induction of SSB by relatively low c oncentrations of glucose oxidase was of a biphasic mode with a fast increas e 2 to 5 min after the addition of glucose oxidase followed by a gradual de crease toward the original base level during the next 35 to 60 min. This re sponse of the cells appears to be based on the activation of already existi ng defense system(s) because it was shown that H2O2 is continuously release d during the reaction time and the inhibition of protein synthesis does not affect the observed pattern. Supplementation of the growth medium with var ious antioxidants resulted in substantial protection only when the agents w ere taken up by the cells. The presence of the intracellular calcium chelat or BAPTA protected the cells from H2O2-induced DNA damage in a dose-depende nt manner. Only at the higher rate of H2O2-generation considerable DNA dama ge was observed in the presence of BAPTA. These results suggest that H2O2, at low concentrations induces DNA damage through intracellular Ca2+-meddate d processes, which lead to DNA strand breaks possibly by endonuclease activ ation. (C) 1999 Elsevier Science Inc.