Induction of 5,6-Ring-saturated Thymine Bases in NIH-3T3 Cells by Phorbol Ester-stimulated Macrophages: Role of Reactive Oxygen Intermediates1

Because oxygen intermediates secreted by inflammatory leu kocytes are postulated to play a role in potentiating carcinogenesis, we investigated the ability of macrophages to induce oxidative DNA damage in eukaryotic cells. Murine macrophages, obtained from sites of inflammation and stimulated with 12-0tetradecanoylphorbol-15-acetate, induced the formation of 5,6ring-saturated thymine bases in the DNA of cocultured NIH-3T3 cells; macrophages or ^-O-tetradecanoylphorboMS-acetate alone did not induce such alterations. Reagent H2O2,at concen trations produced by macrophages in the ambient medium (i.e., ~10~5 M), induced saturated thymines in the target cells in a dose-dependent manner. The reaction between reagent H2O2 and cellular DNA was rapid, reaching maximum levels in 30 min, and similar amounts of saturated thymines were induced at 4° or 37°. The 3T3 targets were able to repair the saturated thymines rapidly (i.e., over 70% of the lesion was removed in 2 hr). Catalase completely inhibited macrophage-mediated induc tion of saturated thymines, although Superoxide dismutase en hanced induction. Taken together, the data indicate that macro phages exposed to phorbol diesters can induce a specific, quan tifiable lesion in the DNA of bystander eukaryotic cells and that reactive oxygen species from the macrophages participate in producing the lesion.