cytochalasin-d and titanium-dioxide

In crystallopathies, crystals or crystalline particles of environmental and metabolic origin deposit within tissues, induce inflammation, injury and cell death and eventually lead to organ-failure. The NLRP3-inflammasome is involved in mediating crystalline particles-induced inflammation, but pathways leading to cell death are still unknown. Here, we have used broad range of intrinsic and extrinsic crystal- or crystalline particle-sizes and shapes, e.g. calcium phosphate, silica, titanium dioxide, cholesterol, calcium oxalate, and monosodium urate. As kidney is commonly affected by crystallopathies, we used human and murine renal tubular cells as a model system. We showed that all of the analysed crystalline particles induce caspase-independent cell death. Deficiency of MLKL, siRNA knockdown of RIPK3, or inhibitors of necroptosis signaling e.g. RIPK-1 inhibitor necrostatin-1s, RIPK3 inhibitor dabrafenib, and MLKL inhibitor necrosulfonamide, partially protected tubular cells from crystalline particles cytotoxicity. Furthermore, we identify phagocytosis of crystalline particles as an upstream event in their cytotoxicity since a phagocytosis inhibitor, cytochalasin D, prevented their cytotoxicity. Taken together, our data confirmed the involvement of necroptosis as one of the pathways leading to cell death in crystallopathies. Our data identified RIPK-1, RIPK3, and MLKL as molecular targets to limit tissue injury and organ failure in crystallopathies.

Topics: Animals; Apoptosis; Calcium Oxalate; Calcium Phosphates; Cell Line; Cholesterol; Crystallization; Cytochalasin D; Epithelial Cells; Gene Expression Regulation; Humans; Imidazoles; Indoles; Inflammasomes; Kidney Tubules; Mice; Necrosis; Oximes; Particle Size; Particulate Matter; Phagocytosis; Primary Cell Culture; Protein Kinases; Receptor-Interacting Protein Serine-Threonine Kinases; Signal Transduction; Silicon Dioxide; Titanium; Uric Acid

Many nanoparticles (NPs) have toxic effects on multiple cell lines. This toxicity is assumed to be related to their accumulation within cells. However, the process of internalization of NPs has not yet been fully characterized. In this study, the cellular uptake, accumulation, and localization of titanium dioxide nanoparticles (TiO2 NPs) in rat (C6) and human (U373) glial cells were analyzed using time-lapse microscopy (TLM) and transmission electron microscopy (TEM). Cytochalasin D (Cyt-D) was used to evaluate whether the internalization process depends of actin reorganization. To determine whether the NP uptake is mediated by phagocytosis or macropinocytosis, nitroblue tetrazolium (NBT) reduction was measured and the 5-(N-ethyl-N-isopropyl)-amiloride was used. Expression of proteins involved with endocytosis and exocytosis such as caveolin-1 (Cav-1) and cysteine string proteins (CSPs) was also determined using flow cytometry. TiO2 NPs were taken up by both cell types, were bound to cellular membranes and were internalized at very short times after exposure (C6, 30 min; U373, 2h). During the uptake process, the formation of pseudopodia and intracellular vesicles was observed, indicating that this process was mediated by endocytosis. No specific localization of TiO2 NPs into particular organelles was found: in contrast, they were primarily localized into large vesicles in the cytoplasm. Internalization of TiO2 NPs was strongly inhibited by Cyt-D in both cells and by amiloride in U373 cells; besides, the observed endocytosis was not associated with NBT reduction in either cell type, indicating that macropinocytosis is the main process of internalization in U373 cells. In addition, increases in the expression of Cav-1 protein and CSPs were observed. In conclusion, glial cells are able to internalize TiO2 NPs by a constitutive endocytic mechanism which may be associated with their strong cytotoxic effect in these cells; therefore, TiO2 NPs internalization and their accumulation in brain cells could be dangerous to human health.

Topics: Actins; Amiloride; Animals; Caveolin 1; Cell Line; Cysteine; Cytochalasin D; Endocytosis; Humans; Metal Nanoparticles; Neuroglia; Rats; Titanium

We investigated the effects of nanosized TiO2 particles on the death of mouse leukemia L1210 cells. TiO2 particles suppressed proliferation and induced cell death, as measured by lactate dehydrogenase (LDH) release into the culture medium. Chromatin condensation, which is typical of the initiation of cell death, was observed in approximately 14% cells cultured with titanium dioxide (TiO2) particles for 12 h. Furthermore, giant DNA fragments of approximately 2 Mbp and high-molecular-weight DNA fragments between 100 kbp and 1 Mbp were observed in cells cultured for 18 h with TiO2 particles. These giant and high-molecular-weight DNA fragments were further degraded into smaller DNA fragments, appearing as DNA ladders. Corresponding to the generation of DNA fragments, caspase-3 activity increased in cells treated with TiO2 particles. TiO2 particle-induced LDH release was not inhibited by cytochalasin D, an inhibitor of endocytosis. These results suggest that nanosized TiO2 particles can induce apoptosis associated with DNA fragmentation and caspase-3 activation and that TiO2 particle-induced apoptosis is not caused by endocytosis but is associated with contact of the particles with the cell surface.

Topics: Animals; Apoptosis; Biocompatible Materials; Caspase 3; Cells, Cultured; Cytochalasin D; DNA Fragmentation; Enzyme Activation; L-Lactate Dehydrogenase; Leukemia L1210; Metal Nanoparticles; Mice; Titanium

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.NR8383 rat lung AM were treated with ultrafine (uf), fine (f) TiO2 or fine crystalline silica (DQ12 quartz). Physico-chemical particle properties were investigated by transmission electron microscopy, elemental analysis and thermogravimetry. Aggregation and agglomeration tendency of the particles were determined in assay-specific suspensions by means of dynamic light scattering.All three particle types were rapidly taken up by AM. DQ12 and ufTiO2 , but not fTiO2 , caused increased extracellular reactive oxygen species (ROS), heme oxygenase 1 (HO-1) mRNA expression and tumor necrosis factor (TNF)-α release. Inducible nitric oxide synthase (iNOS) mRNA expression was increased most strongly by ufTiO2 , while DQ12 exclusively triggered interleukin (IL) 1β release. However, oscillations of intracellular calcium concentration and increased intracellular ROS were observed with all three samples. Uptake inhibition experiments with cytochalasin D, chlorpromazine and a Fcγ receptor II (FcγRII) antibody revealed that the endocytosis of fTiO2 by the macrophages involves actin-dependent phagocytosis and macropinocytosis as well as clathrin-coated pit formation, whereas the uptake of ufTiO2 was dominated by FcγIIR. The uptake of DQ12 was found to be significantly reduced by all three inhibitors. Our findings suggest that the contrasting AM responses to fTiO2 , ufTiO2 and DQ12 relate to differences in the involvement of specific uptake mechanisms.

Topics: Air Pollutants; Animals; Calcium; Cell Line; Cell Survival; Chlorpromazine; Cytochalasin D; Endocytosis; Gene Expression; Heme Oxygenase (Decyclizing); Interleukin-1beta; Macrophage Activation; Macrophages, Alveolar; Nanoparticles; Nitric Oxide Synthase Type II; Particle Size; Rats; Reactive Oxygen Species; Receptors, IgG; Silicon Dioxide; Titanium; Tumor Necrosis Factor-alpha

Fine particles (10(2)- to 10(3)-nm diameter) are potentially potent adjuvants in acquired immune responses but little is known about their interaction with pathogen-associated molecular patterns (PAMPs) and impact upon innate immunity. Here we show that 200-nm-sized, food-grade titanium dioxide avidly binds lipopolysaccharide (LPS) with bridging calcium cations, and the complex induces marked proinflammatory signalling in primary human mononuclear phagocytes. In particular, caspase 1-dependent interleukin-1beta (IL-1beta) secretion was induced at levels far greater than for the sum of the individual components, and without concomitant secretion of modulatory cytokines such as interleukin-1 receptor antagonist or transforming growth factor-beta1 (TGF-beta1). Secondly, the conjugate induced apoptotic-like cell death. These responses were inhibited by blockade of both phagocytosis and scavenger receptor uptake. Specific caspase 1-facilitated IL-1beta secretion and apoptosis following phagocytosis are features of cellular responses to certain invasive, enteric pathogens, and hence induction of these events may be mimicked by fine particle-LPS conjugates. The inadvertent adsorption of PAMPs to ingested, inhaled, or "wear" fine particulate matter provides a further potential mechanism for the proinflammatory nature of fine particles.

Topics: Adaptor Proteins, Signal Transducing; Adjuvants, Immunologic; Adsorption; Adult; Apoptosis; Calcium; Cells, Cultured; Cytochalasin D; Humans; Immunosuppressive Agents; Interleukin-1; Interleukin-6; Leukocytes, Mononuclear; Lipopolysaccharides; Nanoparticles; Particle Size; Phagocytosis; Receptors, Interleukin-1; Titanium; Transforming Growth Factor beta1; Tumor Necrosis Factor-alpha

We sought to establish a quantitative method using flow cytometry to study uptake of environmental particulates by alveolar macrophages (AMs). We used right angle light scatter (RAS) to measure uptake of titanium dioxide, quartz, and diesel particulates. After incubation with TiO2 in vitro, AMs showed dose-dependent increases in both cell-associated particles visualized by microscopy and RAS measured by flow cytometry (e.g., fold increase RAS at 4, 8, 16, 32, and 80 micrograms/ml, respectively, = 2 +/- 0.1, 4.0 +/- 0.5, 5.5 +/- 0.5, 9.1 +/- 2.5, 14.3 +/- 0.9; mean +/- SEM). Similar results were obtained with quartz and diesel particles. A strong correlation was observed between particle load per cell and AM RAS after uptake of fluorescent latex beads or fluorescent TiO2 (coated with BODIPY-BSA) (R2 = 0.984, 0.997, respectively). Using this technique, we found AM uptake of environmental particulates to be substantially greater than that of a panel of myelomonocytic and epithelial cell lines, consistent with their physiologic role in pulmonary defenses. RAS measurements have also identified both calcium-dependent and calcium-independent components in AM interactions with inert particles. Although this technique does not allow precise quantitation of particle number or mass per cell, flow cytometric analysis of relative increases in RAS is a useful tool to study AM interactions with a variety of environmental particulates.

Topics: Animals; Calcium; Cells, Cultured; Cricetinae; Cytochalasin D; Dust; Environmental Pollutants; Flow Cytometry; Light; Macrophages, Alveolar; Petroleum; Phagocytosis; Quartz; Scattering, Radiation; Titanium