BackgroundNanomaterials (NMs) can be fine-tuned in their properties resulting in a high number of variants, each requiring a thorough safety assessment. Grouping and categorization approaches that would reduce the amount of testing are in principle existing for NMs but are still mostly conceptual. One drawback is the limited mechanistic understanding of NM toxicity. Thus, we conducted a multi-omics in vitro study in RLE-6TN rat alveolar epithelial cells involving 12 NMs covering different materials and including a systematic variation of particle size, surface charge and hydrophobicity for SiO2 NMs. Cellular responses were analyzed by global proteomics, targeted metabolomics and SH2 profiling. Results were integrated using Weighted Gene Correlation Network Analysis (WGCNA).ResultsCluster analyses involving all data sets separated Graphene Oxide, TiO2_NM105, SiO2_40 and Phthalocyanine Blue from the other NMs as their cellular responses showed a high degree of similarities, although apical in vivo results may differ. SiO2_7 behaved differently but still induced significant changes. In contrast, the remaining NMs were more similar to untreated controls. WGCNA revealed correlations of specific physico-chemical properties such as agglomerate size and redox potential to cellular responses. A key driver analysis could identify biomolecules being highly correlated to the observed effects, which might be representative biomarker candidates. Key drivers in our study were mainly related to oxidative stress responses and apoptosis.ConclusionsOur multi-omics approach involving proteomics, metabolomics and SH2 profiling proved useful to obtain insights into NMs Mode of Actions. Integrating results allowed for a more robust NM categorization. Moreover, key physico-chemical properties strongly correlating with NM toxicity were identified. Finally, we suggest several key drivers of toxicity that bear the potential to improve future testing and assessment approaches.

BackgroundOxidative stress, a commonly used paradigm to explain nanoparticle (NP)-induced toxicity, results from an imbalance between reactive oxygen species (ROS) generation and detoxification. As one consequence, protein carbonyl levels may become enhanced. Thus, the qualitative and quantitative description of protein carbonylation may be used to characterize how biological systems respond to oxidative stress induced by NPs.MethodsWe investigated a representative panel of 24 NPs including functionalized amorphous silica (6), zirconium dioxide (4), silver (4), titanium dioxide (3), zinc oxide (2), multiwalled carbon nanotubes (3), barium sulfate and boehmite. Surface reactivities of all NPs were studied in a cell-free system by electron spin resonance (ESR). NRK-52E cells were treated with all NPs, analyzed for viability (WST-1 assay) and intracellular ROS production (DCFDA assay). Carbonylated proteins were assessed by 1D and/or 2D immunoblotting and identified by matrix assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF/TOF). In parallel, tissue homogenates from rat lungs intratracheally instilled with silver NPs were studied.ResultsEleven NPs induced elevated levels of carbonylated proteins. This was in good agreement with the surface reactivity of the NPs as obtained by ESR and the reduction in cell viability as assessed by WST-1 assay. By contrast, results obtained by DCFDA assay were deviating. Each NP induced an individual pattern of protein carbonyls on 2D immunoblots. Affected proteins comprised cytoskeletal components, proteins being involved in stress response, or cytoplasmic enzymes of central metabolic pathways such as glycolysis and gluconeogenesis. Furthermore, induction of carbonyls upon silver NP treatment was also verified in rat lung tissue homogenates.ConclusionsAnalysis of protein carbonylation is a versatile and sensitive method to describe NP-induced oxidative stress and, therefore, can be used to identify NPs of concern. Furthermore, detailed information about compromised proteins may aid in classifying NPs according to their mode of action.Electronic supplementary materialThe online version of this article (doi:10.1186/s12989-015-0108-2) contains supplementary material, which is available to authorized users.

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Nanoparticles can be efficient foaming agents. Yet, the detailed mechanisms of foam stabilization by these particles remain unclear. In most cases, the foamability and foam stability of a system have to be determined empirically. We used a multiscale approach to reveal how the microscopic properties of the nanoparticle dispersion are translated into their foaming behavior at the macroscopic scale. As a model system we used silica nanoparticles that were hydrophobized by the in situ adsorption of short-chain alkylamines of chain length C5 to C8. We used fluorescence spectroscopy and electrophoretic mobility measurements to characterize the bulk behavior of the nanoparticles with adsorbed amines. The interfacial behavior was probed by compressing particle monolayers while monitoring the surface tension. The macroscopic foamability and foam stability were evaluated. There are strong correlations between the system properties at all length scales. The most prominent effects are observed at a critical bulk concentration of amines at which the nanoparticles start to aggregate due to hydrophobic interactions. Our study shows how the foam properties are related to the features of the bulk dispersions and to the ordering of particles at the air/water interface. The present results help to understand the surfactant concentration dependent stages of foaming behavior of in situ hydrophobized nanoparticles.

Background: The immense variety and constant development of nanomaterials (NMs) raise the demand for a facilitated risk assessment, for which knowledge on NMs mode of actions (MoAs) is required. For this purpose, a comprehensive data basis is of paramountcy that can be obtained using omics. Furthermore, the establishment of suitable in vitro test systems is indispensable to follow the 3R concept and to master the high number of NMs. In the present study, we aimed at comparing NM effects in vitro and in vivo using a multi-omics approach. We applied an integrated data evaluation strategy based on proteomics and metabolomics to four silica NMs and one titanium dioxide-based NM. For in vitro investigations, rat alveolar epithelial cells (RLE-6TN) and rat alveolar macrophages (NR8383) were treated with different doses of NMs, and the results were compared to effects on rat lungs after shortterm inhalations and instillations at varying doses with and without a recovery period.Results: Since the production of reactive oxygen species (ROS) is described to be a critical biological effect of NMs, and enrichment analyses con rmed oxidative stress as a signi cant effect upon NM treatment in vitro in the present study, we focused on different levels of oxidative stress. Thus, we found opposite changes for proteins and metabolites that are related to the production of reduced glutathione in alveolar epithelial cells and alveolar macrophages, illustrating that NMs MoAs depend on the used model system. Interestingly, in vivo, pathways related to in ammation were affected to a greater extent than oxidative stress responses. Hence, the assignment of the observed effects to the levels of oxidative stress was different in vitro and in vivo as well. However, the overall classi cation of "active" and "passive" NMs was consistent in vitro and in vivo.Conclusions: The consistent classi cation indicates both tested cell lines to be suitable for NM toxicity assessment even though the induced levels of oxidative stress strongly depend on the used model systems. Thus, the here presented results highlight that model systems need to be carefully revised to decipher the extent to which they can replace in vivo testing.

Several reports on amorphous silica nanomaterial (aSiO2 NM) toxicity have been questioning their safety. Herein, we investigated the in vivo pulmonary toxicity of four variants of aSiO2 NM: SiO2_15_Unmod, SiO2_15_Amino, SiO2_7 and SiO2_40. We focused on alterations in lung DNA and protein integrity, and gene expression following single intratracheal instillation in rats. Additionally, a short-term inhalation study (STIS) was carried out for SiO2_7, using TiO2_NM105 as a benchmark NM. In the instillation study, a significant but slight increase in oxidative DNA damage in rats exposed to the highest instilled dose (0.36 mg/rat) of SiO2_15_Amino was observed in the recovery (R) group. Exposure to SiO2_7 or SiO2_40 markedly increased oxidative DNA lesions in rat lung cells of the exposure (E) group at every tested dose. This damage seems to be repaired, since no changes compared to controls were observed in the R groups. In STIS, a significant increase in DNA strand breaks of the lung cells exposed to 0.5 mg/m3 of SiO2_7 or 50 mg/m3 of TiO2_NM105 was observed in both groups. The detected gene expression changes suggest that oxidative stress and/or inflammation pathways are likely implicated in the induction of (oxidative) DNA damage. Overall, all tested aSiO2 NM were not associated with marked in vivo toxicity following instillation or STIS. The genotoxicity findings for SiO2_7 from instillation and STIS are concordant; however, changes in STIS animals were more permanent/difficult to revert.

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