Catrin Albrecht

Inhaled particles and lung cancer. Part A: Mechanisms

Both occupational and environmental exposure to particles is associated with an increased risk of lung cancer. Particles are thought to impact on genotoxicity as well as on cell proliferation via their ability to generate oxidants such as reactive oxygen species (ROS) and reactive nitrogen species (RNS). For mechanistic purposes, one should discriminate between a) the oxidant‐generating properties of particles themselves (i.e., acellular), which are mostly determined by the physicochemical characteristics of the particle surface, and b) the ability of particles to stimulate cellular oxidant generation. Cellular ROS/RNS can be generated by various mechanisms, including particle‐related mitochondrial activation or NAD(P)H‐oxidase enzymes. In addition, since particles can induce an inflammatory response, a further subdivision needs to be made between primary (i.e., particle‐driven) and secondary (i.e., inflammation‐driven) formation of oxidants. Particles may also affect genotoxicity by their ability to carry surface‐adsorbed carcinogenic components into the lung. Each of these pathways can impact on genotoxicity and proliferation, as well as on feedback mechanisms involving DNA repair or apoptosis. Although abundant evidence suggests that ROS/RNS mediate particle‐induced genotoxicity and mutagenesis, little information is available towards the subsequent steps leading to neoplastic changes. Additionally, since most of the proposed molecular mechanisms underlying particle‐related carcinogenesis have been derived from in vitro studies, there is a need for future studies that evaluate the implication of these mechanisms for in vivo lung cancer development. In this respect, transgenic and gene knockout animal models may provide a useful tool. Such studies should also include further assessment of the relative contributions of primary (inflammation‐independent) and secondary (inflammation‐driven) pathways.

Cellular responses to nanoparticles: Target structures and mechanisms

Nanotechnology makes use of the special surface properties of extremely small particles. In this rapidly growing field, many different materials are produced for a multitude of diverse applications. Possible adverse health effects of these materials however are so far scarcely investigated and are therefore a special task of toxicology. Although strategies for risk assessment have been suggested, the authors of the current review emphasize the fact that on the cellular, subcellular and molecular levels, interactions between nanoparticles (NP) and target cells relevant for the induction of possible adverse health effects are poorly understood. On the basis of existing literature, the potentially most relevant cellular target sites of NP as well as the so far known major molecular events specifically induced by these xenobiotics are reviewed. Starting with NP uptake across the cell membrane, mechanisms of generation of reactive oxygen species and the activation of redox-sensitive signalling cascades are described. Besides the cell membrane, mitochondria and cell nucleus are considered as major cell compartments relevant for possible NP-induced toxicity. Finally, an integrated research protocol is proposed to identify fundamental cellular responses to NP in order to complement current toxicological screening strategies with a mechanism-based approach.

Inhaled particles and lung cancer, part B: Paradigms and risk assessment

Poorly soluble particles of low toxicity (PSP), such as CB, TiO2 and coal mine dust, have been demonstrated to cause lung cancer in rodents, being most pronounced in rats. Adequate epidemiologic studies do not clearly indicate increased lung cancer rates in humans exposed to such particles. This has caused controversial positions in regulatory decisions on PSP on different levels. The present review discusses the current paradigms in rodent particle carcinogenicity, i.e., (i) role of particle overload and of persistent inflammation and (ii) fibrosis as an intermediate step in particle‐induced lung cancer with regard to human risk assessment. Fibrosis, which is usually considered a precursor of lung cancer in humans, was not related to lung tumors in an animal study using 6 different particles, each at 3 dosages. Lung tumors after both inhalation and intratracheal instillation of PSP are related to particle surface dose, which forwards hazard assessment at surface‐based nonoverload concentrations and a standard setting using surface as an exposure metric. The scarce data available on humans do not support the overload concept but suggest a role for persistent lung inflammation. Differences in antioxidant protection between different rodent species correlate with susceptibility to PSP‐induced carcinogenicity and support the need for detailed studies on antioxidant response in humans. Apart from such bridging studies, further focus is also needed on surface chemistry and modifications in relation to their adverse biologic effects.

Cytotoxicity and oxidative DNA damage by nanoparticles in human intestinal Caco-2 cells

Abstract The use of engineered nanoparticles in the food sector is anticipated to increase dramatically, whereas their potential hazards for the gastrointestinal tract are still largely unknown. We investigated the cytotoxic and DNA-damaging effects of several types of nanoparticles and fine particles relevant as food additives (TiO2 and SiO2) or for food packaging (ZnO and MgO) as well as carbon black on human intestinal Caco-2 cells. All particles, except for MgO, were cytotoxic (LDH and WST-1 assay). ZnO, and to lesser extent SiO2, induced significant DNA damage (Fpg-comet), while SiO2 and carbon black were the most potent in causing glutathione depletion. DNA damage by TiO2 was found to depend on sample processing conditions. Interestingly, application of different TiO2 and ZnO particles revealed no relation between particle surface area and DNA damage. Our results indicate a potential hazard of several food-related nanoparticles which necessitate investigations on the actual exposure in humans.

Distinctive Toxicity of TiO2 Rutile/Anatase Mixed Phase Nanoparticles on Caco-2 Cells

Titanium dioxide has a long-standing use as a food additive. Micrometric powders are, e.g., applied as whiteners in confectionary or dairy products. Possible hazards of ingested nanometric TiO(2) particles for humans and the potential influence of varying specific surface area (SSA) are currently under discussion. Five TiO(2)-samples were analyzed for purity, crystallinity, primary particle size, SSA, ζ potential, and aggregation/agglomeration. Their potential to induce cytotoxicity, oxidative stress, and DNA damage was evaluated in human intestinal Caco-2 cells. Only anatase-rutile containing samples, in contrast to the pure anatase samples, induced significant LDH leakage or mild DNA damage (Fpg-comet assay). Evaluation of the metabolic competence of the cells (WST-1 assay) revealed a highly significant correlation between the SSA of the anatase samples and cytotoxicity. The anatase/rutile samples showed higher toxicity per unit surface area than the pure anatase powders. However, none of the samples affected cellular markers of oxidative stress. Our findings suggest that both SSA and crystallinity are critical determinants of TiO(2)-toxicity toward intestinal cells.

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 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.

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.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.

The Compatible Solute Ectoine Protects against Nanoparticle-induced Neutrophilic Lung Inflammation

RATIONALE Inflammatory reactions of the airways induced by nanoparticles of occupational and environmental origin contribute to organ-specific and systemic human diseases. Because this kind of exposure in modern societies is often unavoidable, a strategy of molecular prevention on an individual level could help to prevent inflammation-derived secondary diseases. OBJECTIVES To test whether the compatible solute ectoine [(S)-2-methyl-1,4,5,6-tetrahydropyrimidine-4-carboxylic acid], which is known to reduce cell stress effects on a molecular level, prevents nanoparticle-induced lung inflammation. METHODS Inflammatory parameters were studied in Fischer 344 rats treated with model carbon nanoparticles. The molecular effects of ectoin on proinflammatory signal transduction were demonstrated in the rat and in the human system using cultured lung epithelial cells. MEASUREMENTS AND MAIN RESULTS Ectoine, given with or before the nanoparticles, dose-dependently reduced neutrophil inflammation in the lung. This preventive effect was not observed when lung inflammation was induced by bacterial lipopolysaccharide. Analyses of the underlying mode of action revealed that ectoine acted on lung epithelial cells. Ectoine administration inhibited nanoparticle-induced signaling, which is known to be responsible for proinflammatory reactions in rat lung epithelial cells in vitro as well as in vivo. These findings were corroborated and extended in experiments with cultured human bronchial epithelial cells in which ectoine inhibited nanoparticle-triggered cell signaling and IL-8 induction. CONCLUSIONS Because compatible solutes are compliant natural products without known toxic potential, we propose that this group of substances may be used for the prevention of particle-induced airway inflammation in humans.

Influence of simulated gastrointestinal conditions on particle-induced cytotoxicity and interleukin-8 regulation in differentiated and undifferentiated Caco-2 cells

Abstract Novel aspects of engineered nanoparticles offer many advantages for optimising food products and packaging. However, their potential hazards in the gastrointestinal tract require further investigation. We evaluated the toxic and inflammatory potential of two types of particles that might become increasingly relevant to the food industry, namely SiO2 and ZnO. The materials were characterised for their morphology, oxidant generation and hydrodynamic behaviour. Cytotoxicity and interleukin-8 mRNA and protein expression were evaluated in human intestinal Caco-2 cells. Particle pretreatment under simulated gastric and intestinal pH conditions resulted in reduced acellular ROS formation but did not influence cytotoxicity (WST-1 assay) or IL-8 expression. However, the differentiation status of the cells markedly determined the cytotoxic potency of the particles. Further research is needed to determine the in vivo relevance of our current observations regarding the role of particle aggregation and the stage of intestinal epithelial cell differentiation in determining the hazards of ingested particles.

Evaluation of apoptosis induced by nanoparticles and fine particles in RAW 264.7 macrophages: Facts and artefacts

Current hazard characterisation of nanoparticles (NP) is predominantly based on in vitro test systems, being established for small molecules of drugs and chemicals. However, specific physicochemical properties of NP may result in interference with assay components, biomarkers, or detection systems. In the present study, six types of (nano)particles were screened in RAW 264.7 macrophages by common cytotoxicity methods (WST-1, LDH). Our specific focus was on the investigation of apoptosis (analysis of hypodiploid DNA, phosphatidylserine exposure, caspase 3/7 activation, and Cell Death Detection ELISA). Assays were validated by the well-known apoptosis inducer staurosporine. Our results show that ZnO, DQ12 quartz and amorphous silica are cytotoxic with strong indications for apoptotic effects in RAW 264.7 macrophages, whereas toxicity was absent for MgO. For fine as well as ultrafine TiO(2), no apoptotic effects could be detected except for induction of DNA fragmentation. The results of our study demonstrate the necessity to control on a case-by-case basis for assay interference to avoid misinterpretation of specific in vitro test findings. To obtain valid statements on the potential induction of apoptosis by specific NP the measurement of multiple endpoints is a prerequisite.