Contrasting macrophage activation by fine and ultrafine titanium dioxide particles is associated with different uptake mechanisms

Inhalation of (nano)particles may lead to pulmonary inflammation. However, the precise mechanisms of particle uptake and generation of inflammatory mediators by alveolar macrophages (AM) are still poorly understood. The aim of this study was to investigate the interactions between particles and AM and their associated pro-inflammatory effects in relation to particle size and physico-chemical properties

Zinc oxide nanoparticles induce necrosis and apoptosis in macrophages in a p47phox- and Nrf2-independent manner.

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. We have investigated mechanisms of ZnO nanoparticle-induced apoptosis and necrosis in macrophages in relation to their important role in the clearance of inhaled particulates and the regulation of immune responses during inflammation. In the murine macrophage RAW 264.7 cell line, ZnO treatment caused a rapid induction of nuclear condensation, DNA fragmentation, and the formation of hypodiploid DNA nuclei and apoptotic bodies. The involvement of the essential effector caspase-3 in ZnO-mediated apoptosis could be demonstrated by immunocytochemical detection of activated caspase-3 in RAW 264.7 cells. ZnO specifically triggered the intrinsic apoptotic pathway, because Jurkat T lymphocytes deficient in the key mediator caspase-9 were protected 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 p47(phox) 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 p47(phox) or the oxidant responsive transcription factor Nrf2. Taken together, our data demonstrate that ZnO nanoparticles trigger p47(phox) 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

Investigation of the role of ZnO solubilization on the effects in RAW 264.7 cells.

<p>(A) FACS analysis of RAW 264.7 following a 24 h treatment with freshly prepared ZnO nanoparticles (ZnO I, 0 h), with ZnO suspension that were pre-incubated for 24 h at 37°C (ZnO I, 24 h), resuspended pellets of the 24 h pre-incubated suspension (P-ZnO I, 24 h) and the particle free supernatants collected after centrifugation of the pre-incubated suspension at 16.000 g (S-ZnO I, 24 h). All respective suspensions were prepared at 0.7 g/L, equaling the final treatment dose of 80 µg/cm². Staurosporine (STS, 24 h, 0.1 µM) was used as positive control. Data are expressed as percentage of total cell events (n = 2). Detection of Zn using a fluorescent indicator in RAW 264.7 cells treated with control medium (B), treated for 0 h with ZnO particles 80 µg/cm², freshly suspended (C), treated for 4 h with 5 µg/cm² (D) or 80 µg/cm² (E) freshly suspended ZnO particles or treated for 4 h with supernatant (F) as well as pellet (G) of a 80 µg/cm² ZnO suspension after preincubation for 24 h at 37°C. Original magnification 100×.</p

Effects of ZnO exposure in murine bone marrow-derived macrophages of p47^phox−/− versus wt animals.

<p>(A) Superoxide detection via lucigenin-amplified chemiluminescence depicted in arbitrary units (AU). (B) FACS analysis of sideward scatter related granularity to investigate particle uptake (n = 2). (C) Cell viability determined by WST-1 assay (n = 2). (D) Content of hypodiploid cells determined by FACS analysis after 7-AAD staining (n = 2). STS: staurosporine.</p

Cell viability (WST-1 assay) after 4 h of ZnO exposure observed in a mutant Jurkat T-cell line deficient in caspase-9 (JMR) versus their caspase-9-restored counterpart (JMR/C9).

<p>Data of treated cells are related to corresponding control cells (100% activity, n = 3).</p

Cytotoxic effects of a panel of ZnO particles in RAW 264.7 macrophages.

<p>(A) Cell viability determined by WST-1 assay after 4 h treatment with four different ZnO particles at the indicated concentrations. Data are expressed as a percentage of the non-treated control cells, and represent three independent experiments (i.e. n = 3). As positive control, cells were treated for 4 h with 1 µM staurosporine (STS). (B) FACS analysis after 7-AAD staining revealing cells with hypodiploid DNA content after 4 h treatment with the panel of ZnO particles or staurosporine. Data are expressed as percentage of total cell events (n = 3).</p

Fluorescent staining of RAW 264.7 macrophages after 4 h treatment with ZnO or staurosporine (STS) using TUNEL assay.

<p>Representative multichannel images are shown for apoptotic DNA fragmentation (green staining) and corresponding nuclei (blue staining). Original magnification 100×.</p

Representative Scanning Electron Microscopy images of the four ZnO samples used in present study.

<p>Samples were analyzed by a LEO (Zeiss) 1530 Scanning Electron Microscope at a magnification of 10,000× as well as 50,000× (inserts).</p

DLS curves of ZnO suspensions in complete cell culture medium.

<p>(A) Representative data of freshly prepared suspensions of the four ZnO samples. (B) Comparison of freshly suspended ZnO I versus complete suspension or resuspended pellet of ZnO I after 24 h incubation at 37°C. Data were obtained with a Beckmann Coulter Delsa Nano C.</p