Evaluating Toxicity of Chemicals using a Zebrafish Vibration Startle Response Screening System

We developed a simple screening system for the evaluation of neuromuscular and general toxicity in zebrafish embryos. The modular system consists of ...

Karlsruhe Institute of Technology - Campus Nord

Indoor air pollution is associated with increased morbidity and mortality. Specifically, the health impact of emissions from domestic burning of biomass and coal is most relevant and is estimated to contribute to over 4 million premature deaths per year worldwide. Wood is the main fuel source for biomass combustion and the shift towards renewable energy sources will further increase emissions from wood combustion even in developed countries. However, little is known about the constituents of wood smoke and biological mechanisms that are responsible for adverse health effects. We exposed A549 lung epithelial cells to collected wood smoke particles and found an increase in cellular reactive oxygen species as well as a response to bioavailable polycyclic aromatic hydrocarbons. In contrast, cell vitality and regulation of the pro-inflammatory cytokine interleukin-8 were not affected. Using a candidate approach, we could recapitulate WSP toxicity by the combined actions of its constituents soot, metals and PAHs. The soot fraction and metals were found to be the most important factors for ROS formation, whereas the PAH response can be mimicked by the model PAH benzo[a]pyrene. Strikingly, PAHs adsorbed to WSPs were even more potent in activating target gene expression than B[a]P individually applied in suspension. As PAHs initiate multiple adverse outcome pathways and are prominent carcinogens, their role as key pollutants in wood smoke and its health effects warrants further investigation. The presented results suggest that each of the investigated constituents soot, metals and PAHs are major contributors to WSP toxicity. Mitigation strategies to prevent adverse health effects of wood combustion should therefore not only aim at reducing the emitted soot and PAHs but also the metal content, through the use of more efficient combustion appliances, and particle precipitation techniques, respectively.

Toxicity of wood smoke particles in human A549 lung epithelial cells: the role of PAHs, soot and zinc.

Although the technological and economic benefits of engineered nanomaterials are obvious, concerns have been raised about adverse effects if such material is inhaled, ingested, applied to the skin or even released into the environment. Here we studied the cytotoxic effects of the most abundant nanomaterial, silica nanoparticles (SiO-NPs), in murine RAW264.7 macrophages. SiO-NPs dose-dependently induce membrane leakage and cell death without obvious involvement of reactive oxygen species. Interestingly, at low concentrations SiO-NPs trigger autophagy, evidenced by morphological and biochemical hallmarks such as autophagolysosomes or increased levels of LC3-II, which serves to protect cells from cytotoxicity. Hence SiO-NPs initiate an adaptive stress response which dependent on dose serve to balance survival and death and ultimately dictates the cellular fate.

Autophagy induced by silica nanoparticles protects RAW264.7 macrophages from cell death.

Nanostructured silica particles are commonly used in biomedical and biotechnical fields, as well as, in cosmetics and food industry. Thus, their environmental and health impacts are of great interest and effects after oral uptake are only rarely investigated. In the present study, the toxicological effects of commercially available nano-scaled silica with a nominal primary diameter of 12 nm were investigated on the human gastric carcinoma cell line GXF251L. Besides the analysis of cytotoxic and proliferative effects and the comparison with effects of particles with a nominal primary diameter of 200 nm, emphasis was also given to their influence on the cellular epidermal growth factor receptor (EGFR) and mitogen-activated protein kinases (MAPK) signaling pathways-both of them deeply involved in the regulation of cellular processes like cell cycle progression, differentiation or proliferation. The investigated silica nanoparticles (NPs) were found to stimulate cell proliferation as measured by microscopy and the sulforhodamine B assay. In accordance, the nuclear level of the proliferation marker Ki-67 was enhanced in a concentration-dependent manner. At high particle concentrations also necrosis was induced. Finally, silica NPs affected the EGFR and MAPK pathways at various levels dependent on concentration and time. However, classical activation of the EGFR, to be reflected by enhanced levels of phosphorylation, could be excluded as major trigger of the proliferative stimulus. After 45 min of incubation the level of phosphorylated EGFR did not increase, whereas enhanced levels of total EGFR protein were observed. These results indicate interference with the complex homeostasis of the EGFR protein, whereby up to 24 h no impact on the transcription level was detected. In addition, downstream on the level of the MAP kinases ERK1/2 short term incubation appeared to affect total protein levels without clear increase in phosphorylation. Depending on the concentration range, enhanced levels of ERK1/2 phosphorylation were only observed after 24 h of incubation. Taken together, the present study demonstrates the potential of the tested silica particles to enhance the growth of gastric carcinoma cells. Although interference with the EGFR/MAPK cascade is observed, additional mechanisms are likely to be involved in the onset of the proliferative stimulus.

Amorphous Silica Particles Relevant in Food Industry Influence Cellular Growth and Associated Signaling Pathways in Human Gastric Carcinoma Cells.

Manufactured nanomaterials (MNMs) selected from a library of over 120 different MNMs with varied compositions, sizes, and surface coatings were tested by four different laboratories for toxicity by high-throughput/-content (HT/C) techniques. The selected particles comprise 14 MNMs composed of CeO2, Ag, TiO2, ZnO and SiO2 with different coatings and surface characteristics at varying concentrations. The MNMs were tested in different mammalian cell lines at concentrations between 0.5 and 250 µg/mL to link physical-chemical properties to multiple adverse effects. The cell lines are derived from relevant organs such as liver, lung, colon and the immune system. Endpoints such as viable cell count, cell membrane permeability, apoptotic cell death, mitochondrial membrane potential, lysosomal acidification and steatosis have been studied. Soluble MNMs, Ag and ZnO, were toxic in all cell types. TiO2 and SiO2 MNMs also triggered toxicity in some, but not all, cell types and the cell type-specific effects were influenced by the specific coating and surface modification. CeO2 MNMs were nearly ineffective in our test systems. Differentiated liver cells appear to be most sensitive to MNMs, Whereas most of the investigated MNMs showed no acute toxicity, it became clear that some show adverse effects dependent on the assay and cell line. Hence, it is advised that future nanosafety studies utilise a multi-parametric approach such as HT/C screening to avoid missing signs of toxicity. Furthermore, some of the cell type-specific effects should be followed up in more detail and might also provide an incentive to address potential adverse effects in vivo in the relevant organ.

Microscopy-based high-throughput assays enable multi-parametric analysis to assess adverse effects of nanomaterials in various cell lines.

A central challenge for the safe design of nanomaterials (NMs) is the inherent variability of NM properties, both as produced and as they interact with and evolve in, their surroundings. This has led to uncertainty in the literature regarding whether the biological and toxicological effects reported for NMs are related to specific NM properties themselves, or rather to the presence of impurities or physical effects such as agglomeration of particles. Thus, there is a strong need for systematic evaluation of the synthesis and processing parameters that lead to potential variability of different NM batches and the reproducible production of commonly utilized NMs. The work described here represents over three years of effort across 14 European laboratories to assess the reproducibility of nanoparticle properties produced by the same and modified synthesis routes for four of the OECD priority NMs (silica dioxide, zinc oxide, cerium dioxide and titanium dioxide) as well as amine-modified polystyrene NMs, which are frequently employed as positive controls for nanotoxicity studies. For 46 different batches of the selected NMs, all physicochemical descriptors as prioritized by the OECD have been fully characterized. The study represents the most complete assessment of NMs batch-to-batch variability performed to date and provides numerous important insights into the potential sources of variability of NMs and how these might be reduced.

Characterization of Nanoparticle Batch-To-Batch Variability.

Engineered amorphous silica nanoparticles (nanosilica) are widely used in industry yet can induce adverse effects, which might be classified according to the oxidative stress model. However, the underlying mechanisms as well as the potential interactions of the three postulated different tiers of toxicity-i.e. oxidative-, pro-inflammatory- and cytotoxic-stress response-are poorly understood. As macrophages are primary targets of nanoparticles, we used several macrophage models, primarily murine RAW264.7 macrophages, and monitored pro-inflammatory and anti-oxidative reactions as well as cytotoxicity in response to nanosilica at max. 50 µg/mL. Special attention was given to the activation of mitogen-activated protein kinases (MAPKs) as potential regulators of the cellular stress response. Indeed, according to the oxidative stress model, also nanosilica elicits an, albeit modest, anti-oxidative response as well as pronounced pro-inflammatory reactions and cytotoxicity in macrophages. Interestingly however, these three tiers of toxicity seem to operate separately of each other for nanosilica. Specifically, impeding the anti-oxidative response by scavenging of reactive oxygen species does not prevent the pro-inflammatory and cytotoxic response. Furthermore, blocking the pro-inflammatory response by inhibition of MAPKs does not impair cell death. As hazard assessment has been guided by the prevailing assumption of a dose-dependent coupling of sequential tiers of toxicity, identification of critical physico-chemical parameters to assist the safe-by-design concept should be enabled by simply monitoring one of the toxicity read-outs. Our results indicate a more complex scenario in the case of nanosilica, which triggers independent pleiotropic effects possibly also related to different material properties and primary cellular targets.

Revisiting the stress paradigm for silica nanoparticles: decoupling of the anti-oxidative defense, pro-inflammatory response and cytotoxicity.

Engineered amorphous silica nanoparticles (nanosilica) are one of the most abundant nanomaterials and are widely used in industry. Furthermore, novel nanosilica materials are promising theranostic tools for biomedicine. However, hazardous effects of nanosilica especially after inhalation into the lung have been documented. Therefore, the safe development of nanosilica materials urgently requires predictive assays to monitor toxicity. Here, we further investigate the impact of the protein corona on the biological activity of two different types of nanosilica (colloidal and pyrogenic) in lung cells. As previously described, adsorption of serum proteins to the nanosilica surface suppresses cytotoxicity in macrophages and lung epithelial cells. As the increase of pro-inflammatory mediators is a hallmark of inflammation in the lung upon nanosilica exposure, we studied the potential coupling of the cytotoxic and pro-inflammatory response in A549 human lung epithelial cells and RAW264.7 mouse macrophages. Indeed, cytotoxicity precedes the onset of pro-inflammatory gene expression and cytokine release as exemplified for IL-8 in A549 cells and TNF-alpha in RAW264.7 macrophages after exposure to 0-100 µg/mL nanosilica in medium without serum. Formation of a protein corona not only inhibited cellular toxicity, but also the pro-inflammatory response. Of note, uptake of nanosilica into cells was negligible in the absence, but enhanced in the presence of a protein corona. Hence, the prevailing explanation that the protein corona simply interferes with cellular uptake thus preventing adverse effects needs to be revisited. In conclusion, for the reliable prediction of adverse effects of nanosilica in the lung, in vitro assays should be performed in media not complemented with complete serum. However, in case of different exposure routes, e.g., injection into the blood stream as intended for biomedicine, the protein corona prevents acute toxic actions of nanosilica.

The protein corona suppresses the cytotoxic and pro-inflammatory response in lung epithelial cells and macrophages upon exposure to nanosilica.

Several in vitro studies have suggested that silica nanoparticles (NPs) might induce adverse effects in gut cells. Here, we used the human colon cancer epithelial cell line HCT116 to study the potential cytotoxic effects of ingested silica NPs in the presence or absence of serum. Furthermore, we evaluated different physico-chemical parameters important for the assessment of nanoparticle safety, including primary particle size (12, 70, 200, and 500 nm) and surface modification (-NH and -COOH). Silica NPs triggered cytotoxicity, as evidenced by reduced metabolism and enhanced membrane leakage. Automated microscopy revealed that the silica NPs promoted apoptosis and necrosis proportional to the administered specific surface area dose. Cytotoxicity of silica NPs was suppressed by increasing amount of serum and surface modification. Furthermore, inhibition of caspases partially prevented silica NP-induced cytotoxicity. In order to investigate the role of specific cell death pathways in more detail, we used isogenic derivatives of HCT116 cells which lack the pro-apoptotic proteins p53 or BAX. In contrast to the anticancer drug cisplatin, silica NPs induced cell death independent of the p53-BAX axis. In conclusion, silica NPs initiated cell death in colon cancer cells dependent on the specific surface area and presence of serum. Further studies in vivo are warranted to address potential cytotoxic actions in the gut epithelium. The unintended toxicity of silica NPs as observed here could also be beneficial. As loss of p53 in colon cancer cells contributes to resistance against anticancer drugs, and thus to reoccurrence of colon cancer, targeted delivery of silica NPs could be envisioned to also deplete p53 deficient tumor cells.

Silica Nanoparticles Provoke Cell Death Independent of p53 and BAX in Human Colon Cancer Cells.

Epidemiological studies identified air pollution as one of the prime causes for human morbidity and mortality, due to harmful effects mainly on the cardiovascular and respiratory systems. Damage to the lung leads to several severe diseases such as fibrosis, chronic obstructive pulmonary disease and cancer. Noxious environmental aerosols are comprised of a gas and particulate phase representing highly complex chemical mixtures composed of myriads of compounds. Although some critical pollutants, foremost particulate matter (PM), could be linked to adverse health effects, a comprehensive understanding of relevant biological mechanisms and detrimental aerosol constituents is still lacking. Here, we employed a systems toxicology approach focusing on wood combustion, an important source for air pollution, and demonstrate a key role of the gas phase, specifically carbonyls, in driving adverse effects. Transcriptional profiling and biochemical analysis of human lung cells exposed at the air-liquid-interface determined DNA damage and stress response, as well as perturbation of cellular metabolism, as major key events. Connectivity mapping revealed a high similarity of gene expression signatures induced by wood smoke and agents prompting DNA-protein crosslinks (DPCs). Indeed, various gaseous aldehydes were detected in wood smoke, which promote DPCs, initiate similar genomic responses and are responsible for DNA damage provoked by wood smoke. Hence, systems toxicology enables the discovery of critical constituents of complex mixtures i.e. aerosols and highlights the role of carbonyls on top of particulate matter as an important health hazard.

Carsten Weiss

Institute of Biological and Chemical Systems - Biological Information Processing

Karlsruhe Institute of Technology - Campus Nord

Evaluating Toxicity of Chemicals using a Zebrafish Vibration Startle Response Screening System

Carsten Weiss

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Institute of Biological and Chemical Systems - Biological Information Processing

Karlsruhe Institute of Technology - Campus Nord

Evaluating Toxicity of Chemicals using a Zebrafish Vibration Startle Response Screening System

Institute of Biological and Chemical Systems - Biological Information Processing