Peroxide in Human Cells1

activity in the same study (14), possibly because the stimulating effect in vivo may be a short-term phenomenon (14) or perhaps because of an intracellular inactivation of SOD by ROS (16). More directly, Clutton and coworkers (17) have reported that the progeny of murine bone marrow cells that were exposed to high-linear energy transfer neutrons showed an enhanced ability to oxidize intracellular DCFH, which was associated both with an increase in 8-hydroxy-2-deoxygua nine, an index of DNA oxidative base damage, and with DNA fragmentation. Further, we (18) recently found that exposure of se rum-containing culture medium to a particles results in the generation of a short-lived SCE-inducing factor and that low dose a-irradiation of human cells results in the generation of a more persistent, heat labile, cell-derived, SCE-inducing factor. These factors can induce excessive SCEs in normal fibroblasts to the same extent as that observed when the cells are directly irradiated. In the context of ROS, both of the SCE-inducing responses to the factors were found to be inhibitable by SOD. Most simply, these latter observations are also consistent with the possibility that a particles can damage DNA indirectly via a mechanism or mechanisms involving cellular gener ation of ROS. Here, we investigated the hypothesis that a particles can induce the cellular production of O2@ and H2O2 by assessing intracellular O2@ and H2O2 with flow cytometry (19â€"21).We found that the intracel lular production of O2@ and H202 is indeed increased following exposure to low doses of a particles. Additionally, we report that such increases in intracellular ROS do not require direct nuclear or cellular/ cytoplasmic hits by a particles and that the ROS response can be induced by extracellular factors in a manner similar to which we previously observed with the induction of excessive SCEs (18). ABSTRACT The mechanism(s)by whichhigh-linearenergytransfera particles,like those emitted by inhaled radon and radon daughters, cause lung cancer has not been elucidated. Conceivably, DNAdamage that is induced by a particles may be mediated by the metabolic generation of reactive oxygen species (ROS), in addition to direct a particle.DNA interactions and hydroxyl radical-DNA interactions. Using normal human lung fibroblasts, we investigated the hypothesis that densely ionizing a particles may Induce the intracellular generation of superoxide (°2@) and hydrogen peroxide (H2O@). Ethidium bromide and 2',7'-dichlorofluorescein, fluo. rescent products of the membrane-permeable dyes hydroethidine and 2',7'-dichlorofluorescin diacetate, respectively, were used to monitor the intracellular production of O2@and H202, respectively, by flow cytom. etry. Compared to sham.irradiated cells, fibroblasts that were exposed to a particles (0.4â€"19 cGy) had significant Increasesin intracellular °2 production, along with concomitant increases in H202 production. Fur ther analyses suggest that the plasma membrane.bound NADPH-oxidase is primarily responsible for this increased intracellular generation of ROS and that the ROS response does not require direct nuclear or cellular “hits” by the a particles. In this latter regard, we additionally report that unirradiated cells also show the ROS response when they are incubated with serum-containing culture medium that has been exposed to a parti. des or when they are incubated with supernatants from a-irradiated cells. Our overall results support the possibility that a particles, at least in part, may mediate their DNA-damagingeffects indirectly via a ROS-related mechanism.