Establishment of a human system that generates O2- and induces 8-hydroxydeoxyguanosine, typical of oxidative DNA damage, by a tumor promotor

Abstract

We have established a system in which a human cell line generated reactive oxygen species (ROS), using a dimethyl sulfoxide-differentiated promyelocytic leukemia cell line HL60 (DMSO-HL60), which has characteristics similar to those of human neutrophils. DMSO-HL60 generated O2- upon stimulation with a tumor promoter, phorbol myristate acetate (PMA). O2- generation, determined as O2- release from the PMA-stimulated cells by the reduction of cytochrome c, was dependent on the dose of PMA and reached almost maximal with 2.0 nM PMA. PMA dose-dependently increased 8-hydroxydeoxyguanosine (8OHdG) in DMSO-HL60, typical of mutagenic oxidative DNA damage. The 8OHdG level, determined by electrochemical detection with high performance liquid chromatography, also became almost maximal with 2.0 nM PMA. The amount of O2- generation and that of the 8OHdG induction by PMA in human neutrophils were similar to those in DMSO-HL60. Superoxide dismutase inhibited the 8OHdG induction by about 60%, whereas catalase, deferoxamine, or thiols inhibited it almost completely. Dilution of PMA-stimulated DMSO-HL60 decreased the concentration of ROS releasing to the media from the cells. However, it did not decrease the ROS generation per cell or the 8OHdG induction. The addition of H2O2 to unstimulated DMSO-HL60 did not increase the 8OHdG level. These findings indicate that DMSO-HL60 could be used as a substitute for human neutrophils, that the concentration of ROS is not the only determinant for the 8OHdG induction, but that it requires both acquisition of susceptibility to ROS by reduction of iron by O2- and formation of H2O2, and that ROS increase 8OHdG by attacking the ROS-generating cell.