In view of the steadily increasing use of zinc oxide nanoparticles in various industrial and consumer applications, toxicological investigations to evaluate their safety are highly justified. against ZnO-mediated toxicity whereas reconstituted cells were not. ZnO also caused DNA strand breakage and oxidative DNA damage in the RAW 264.7 cells as well as p47phox NADPH oxidase-dependent superoxide generation in bone marrow-derived macrophages. However, ZnO-induced cell death was not affected in bone marrow-derived macrophages of mice deficient in p47phox or the oxidant responsive transcription factor Nrf2. Taken together, our data demonstrate that ZnO nanoparticles trigger p47phox NADPH oxidase-mediated ROS formation in macrophages, but that this is dispensable for caspase-9/3-mediated apoptosis. Execution of apoptotic cell death by ZnO nanoparticles appears to be NADPH oxidase and Nrf2-independent but rather triggered by alternative routes. Introduction Nanotechnology is one of the key technologies of the current and upcoming decades, creating an enormous number of novel marketing potentials. Especially metallic nanoparticles offer great industrial opportunities due to their unique properties. Among these are zinc oxide nanoparticles (ZnO NP), which are produced in high tonnage and utilized in many commercial products. Because of their excellent UV-adsorbing properties and concurrent transparency for visible light, ZnO NP have found their use as efficient UV-protectors in cosmetics like sunscreens as well as in paints or finishing materials of building storefronts [1], [2]. Antibacterial properties of this material are used in household products like toothpaste or in food-packaging materials [3], [4]. In the fields of biotechnology and nanomedicine ZnO-based biosensors and biomedical nanomaterials containing ZnO are being developed for cancer treatment applications and improved drug delivery [5], [6]. The broad applicability of ZnO nanoparticles implies human exposure via different body entrance routes, including inhalation and ingestion. Macrophages are strategically located throughout the body tissues and play a central role in the defense against foreign material, dead cells and debris; these processes are implicated in both protective and adverse functions of macrophages in the regulation of the immune response in various pathogenic processes including inflammation and fibrosis [7]. Regarding particulate matter, macrophages are the most important cell type for uptake and clearance processes [8], [9], [10]. There is evidence that mononuclear cells, presumably the resident alveolar macrophages, mediate metal-related parenchymal disorders in occupational settings, such as metal fume fever which may result from inhalation of ZnO particles [11]. Investigations with crystalline silica dust have revealed a clear association between particle-induced apoptotic processes and the development of lung fibrosis [12]. Several recent studies have shown considerable cytotoxicity of ZnO NP to specific cell types, microorganisms and models [10], [13], [14], [15], [16], [17]. However, there are still a lot of controversies regarding the underlying pathways implicated in ZnO-induced cell death. This includes the impact of specific physicochemical properties of this material, like particle size and dissolution as well as the formation of reactive oxygen species (ROS) and Hpt the associated 13602-53-4 supplier oxidative stress involving induction of lipid peroxidation and oxidative DNA damage [13], [14], [15], [18], [19], [20]. In professional phagocytes, such as macrophages and neutrophils, the dominant source of ROS is the classic nicotinamide adenine dinucleotide phosphate (NADPH) oxidase enzyme complex NOX2. Activation of this complex involves the recruitment and assembly of multiple cytosolic subunits including p47phox, p67phox and p40phox with its membrane-bound subcomplex consisting of gp91phox, p22phox and Rac and results in the rapid generation of large amounts of superoxide anion (O2?) [21]. The NOX2-mediated oxidative burst represents a hallmark of the innate host defense to invading microorganisms. However, it is also strongly implicated in the adverse pulmonary effects of well-known particulate toxicants including asbestos and respirable crystalline silica dust [22], [23], [24]. Cells typically accomplish oxidative stress with the induction of antioxidant and detoxification enzymes mediated by the transcription factor nuclear factor erythroid-derived 2 related factor 2 (Nrf2) and hence may counteract the effects of necrosis and apoptosis-inducing triggers [25], [26], [27]. During oxidative stress, Nrf2 is released from its cytosolic repressor Keap1 and subsequently translocates into the nucleus where it binds to antioxidant response elements (ARE) residing within the promoter regions of many antioxidant and phase II genes [28]. The establishment of Nrf2 knockout mouse models has provided major support for the protective role of this pathway in ROS mediated cell death and diseases [29], [30] The formation of ROS and associated induction of oxidative stress have been strongly linked to apoptosis in macrophages [31]. Apoptotic pathways induced by ROS-mediated DNA damage are fundamentally governed by mitochondria due to the release of apoptogenic factors out of the intermembrane space. The release of 13602-53-4 supplier cytochrome c initiates the formation of the apoptosome, composed of Apaf-1 and the initiator caspase-9, resulting in caspase-9 activation. This complex in turn activates the executioner caspase-3, -6 and -7. Cleavage of their specific substrates leads to 13602-53-4 supplier the typical apoptotic cellular.