CHEMILUMINESCENCE AND SUPEROXIDE PRODUCTION IN ACANTHAMOEBA-CASTELLANII - FREE-RADICALS GENERATED DURING OXIDATIVE STRESS

The amoeba Acanthamoeba castellanii generated both luminol- and lucigenin-enhanced chemiluminescence upon addition of the respiratory inhibitor sodium cyanide, but not upon the addition of sodium azide. Photon emission in the presence of lucigenin was three- to fourfold greater than that measured in the presence of luminol, but both forms of chemiluminescence were strictly dependent upon the presence of O2, indicating the requirement for oxidative reactions in these processes. Lucigenin-chemiluminescence measured during the phagocytosis of latex beads under identical conditions was, however, barely detectable above background levels. Cyanide similarly induced the formation of O2-, as indicated by the stimulation of superoxide-dismutase-inhibitable cytochrome c reduction, and the rate of production was similar to that previously observed during phagocytosis of latex particles or yeasts by these cells. Thus, in view of the similar rates of O2- production during cyanide exposure or phagocytosis, but disparate rates of lucigenin-chemiluminescence, these two treatments must activate different molecular processes leading to reactive oxidant production. The rates of cyanide-stimulated lucigenin-chemiluminescence were directly proportional to the O2 tensions in the medium from 0 to 300-mu-M, indicating that the rates of free-radical-generating reactions were directly related to the oxygen tensions in the environment. The superoxide dismutase inhibitor diethyldithiocarbamate similarly stimulated lucigenin-chemiluminescence, with photon emission again being dependent upon O2 tensions in the range 0-320-mu-M. A mechanism by which cells may limit O2--generating reactions, and so reduce damaging free radical reactions, was observed when anaerobic suspensions were re-aerated. These data indicate that oxidative stress and phagocytosis provide two intracellular sources of free-radical generating reactions in these cells.