Targeting NADPH-oxidase by reactive oxygen species reveals an initial sensitive step in the assembly process.

NADPH-oxidase (Nox) is a highly regulated dynamic complex comprising membrane and cytosolic proteins and is the major source of nonmitochondrial cellular reactive oxygen species (ROS). In phagocyte cells, in which ROS are produced in huge amounts, Nox is "naturally" assailed by the action of its own ROS. We have subjected each individual component of Nox or the whole complex at various times during the assembly process either to oxygen free radicals produced by radiolysis or to hydrogen peroxide. Membrane components presented the highest irradiation sensitivity. Irradiation of p67(phox) drastically decreased its interaction with arachidonic acid and destabilized the [p47(phox)-p67(phox)] complex. When the system was irradiated during its assembly process, we could identify an initial irradiation-sensitive phase followed by a resistant form when the complex was assembled.

Effect of UV irradiation on functional activity of donor blood neutrophils.

Activation of NADPH-oxidase enzymatic complex was observed after UV irradiation of the blood and neutrophil suspension in a dose of 151 J/m(2). UV irradiation in doses of 75.5 and 151.0 J/m(2) corrected myeloperoxidase activity and intensity of LPO processes in donor blood.

Fifty-hertz magnetic fields induce free radical formation in mouse bone marrow-derived promonocytes and macrophages.

Our findings show a significant increase of free radical production after exposure to 50 Hz electromagnetic fields at a flux density of 1 mT to mouse bone marrow-derived (MBM) promonocytes and macrophages, indicating the cell-activating capacity of extremely low frequency magnetic fields (ELF-MF). We demonstrate that after exposure to ELF-MF mainly superoxide anion radicals were produced, both in MBM macrophages (33%) and also in their precursor cells (24%). To elucidate whether NADPH- or NADH-oxidase functions are target proteins for MF interaction, the flavoprotein inhibitor diphenyleneiodonium chloride (DPI) was used. MF-induced free radical production was not inhibited by DPI, whereas tetradecanoylphorbolacetate (TPA)-induced free radical production was diminished by about 70%. TPA is known to induce a direct activation of NADPH-oxidase through the PKC pathway. Since DPI lacks an inhibitory effect in MF-exposed MBM cells, we suggest that 50 Hz MF stimulates the NADH-oxidase pathway to produce superoxide anion radicals, but not the NADPH pathway. Furthermore, we showed an oscillation (1-10 days) in superoxide anion radical release in mouse macrophages, indicating a cyclic pattern of NADH-oxidase activity.

Enhancement of O2- production from resident peritoneal macrophages by low-dose in vivo gamma-irradiation.

Superoxide anion (O2-) production after very low-dose in vivo irradiation (4 cGy) was examined in resident peritoneal macrophages. The level of production rapidly increased following treatment with the PKC activator, phorbol 12-myristate 13-acetate (PMA), but no further enhancement by low-dose in vivo irradiation was observed. On the other hand, treatment with zymosan A gradually induced O2- production, which was further increased in low-dose in vivo irradiated macrophages. The amounts of phagocytosis of zymosan A were not changed by in vivo irradiation. This indicated that the enhancement of O2- production was not due to an increase in phagocytotic activity by low-dose in vivo irradiation. Our results show that low-dose in vivo irradiation induces production of reactive oxygen species by macrophages, not only nitric oxide as reported in our previous paper but also O2-. This may contribute to the increase of cytolytic activity of macrophages after low-dose in vivo irradiation.