Differentially Induced Autophagy by Engineered Nanomaterial Treatment Has an Impact on Cellular Homeostasis and Cytotoxicity.

Considering the increasing production of engineered nanomaterials (ENMs), new approach methodologies (NAMs) are essential for safe-by-design approaches and risk assessment. Our aim was to enhance screening strategies with a focus on reactivity-triggered toxicities. We applied in vitro tests to 10 selected benchmark ENMs in two cell models, lung epithelial A549 and differentiated THP-1 macrophage-like cells. Previously, we categorized ENMs based on surface reactivity. Here we elucidated their reactivity-triggered cytotoxicity and mode of action using the WST-1 assay (metabolic activity), LDH assay (cell membrane integrity), autophagosome detection, and proteomics. Nonreactive SiO2 NM-200 showed no significant impact on cell viability. Conversely, highly reactive CuO and ZnO (NM-110 and NM-111) disrupted cell homeostasis. Interestingly, moderately reactive TiO2 (NM-101 and NM-105) and CeO2 (NM-211 and NM-212), apparently without an adverse effect, induced autophagosome formation, evidencing autophagy as a defensive mechanism. Our improved in vitro testing strategy, combined with state-of-the-art reactivity information, screens ENMs for potential reactivity-triggered toxicity.

Towards characterization of cell culture conditions for reliable proteomic analysis: in vitro studies on A549, differentiated THP-1, and NR8383 cell lines.

Proteomic investigations result in high dimensional datasets, but integration or comparison of different studies is hampered by high variances due to different experimental setups. In addition, cell culture conditions can have a huge impact on the outcome. This study systematically investigates the impact of experimental parameters on the proteomic profiles of commonly used cell lines-A549, differentiated THP-1 macrophage-like cells, and NR8383-for toxicity studies. The work focuses on analyzing the influence at the proteome level of cell culture setup involving different vessels, cell passage numbers, and post-differentiation harvesting time, aiming to improve the reliability of proteomic analyses for hazard assessment. Mass-spectrometry-based proteomics was utilized for accurate protein quantification by means of a label-free approach. Our results showed that significant proteome variations occur when cells are cultivated under different setups. Further analysis of these variations revealed their association to specific cellular pathways related to protein misfolding, oxidative stress, and proteasome activity. Conversely, the influence of cell passage numbers on the proteome is minor, suggesting a reliable range for conducting reproducible biological replicates. Notable, substantial proteome alterations occur over-time post-differentiation of dTHP-1 cells, particularly impacting pathways crucial for macrophage function. This finding is key for the interpretation of experimental results. These results highlight the need for standardized culture conditions in proteomic-based evaluations of treatment effects to ensure reliable results, a prerequisite for achieving regulatory acceptance of proteomics data.

A comparative proteomics analysis of four contact allergens in THP-1 cells shows distinct alterations in key metabolic pathways.

Allergic contact dermatitis (ACD) is the predominant form of immunotoxicity in humans. The sensitizing potential of chemicals can be assessed in vitro. However, a better mechanistic understanding could improve the current OECD-validated test battery. The aim of this study was to get insights into toxicity mechanisms of four contact allergens, p-benzoquinone (BQ), 2,4-dinitrochlorobenzene (DNCB), p-nitrobenzyl bromide (NBB) and NiSO4, by analyzing differential proteome alterations in THP-1 cells using two common proteomics workflows, stable isotope labeling by amino acids in cell culture (SILAC) and label-free quantification (LFQ). Here, SILAC was found to deliver more robust results. Overall, the four allergens induced similar responses in THP-1 cells, which underwent profound metabolic reprogramming, including a striking upregulation of the TCA cycle accompanied by pronounced induction of the Nrf2 oxidative stress response pathway. The magnitude of induction varied between the allergens with DNCB and NBB being most potent. A considerable overlap between transcriptome-based signatures of the GARD assay and the proteins identified in our study was found. When comparing the results of this study to a previous proteomics study in human primary monocyte-derived dendritic cells, we found a rather low share in regulated proteins. However, on pathway level, the overlap was high, indicating that affected pathways rather than single proteins are more eligible to investigate proteomic changes induced by contact allergens. Overall, this study confirms the potential of proteomics to obtain a profound mechanistic understanding, which may help improving existing in vitro assays for skin sensitization.

The application of existing genotoxicity methodologies for grouping of nanomaterials: towards an integrated approach to testing and assessment.

The incorporation of nanomaterials (NMs) in consumer products has proven to be highly valuable in many sectors. Unfortunately, however, the same nano specific physicochemical properties, which make these material attractive, might also contribute to hazards for people exposed to these materials. The physicochemical properties of NMs will impact their interaction with biological surroundings and influence their fate and their potential adverse effects such as genotoxicity. Due to the large and expanding number of NMs produced, their availability in different nanoforms (NFs) and their utilization in various formats, it is impossible for risk assessment to be conducted on an individual NF basis. Alternative methods, such as grouping are needed for streamlining hazard assessment. The GRACIOUS Framework provides a logical and science evidenced approach to group similar NFs, allowing read-across of hazard information from source NFs (or non-NFs) with adequate hazard data to target NFs that lack such data. Here, we propose a simple three-tiered testing strategy to gather evidence to determine whether different NFs are sufficiently similar with respect to their potential to induce genotoxicity, in order to be grouped. The tiered testing strategy includes simple in vitro models as well as a number of alternative more complex multi-cellular in vitro models to allow for a better understanding of secondary NM-induced DNA damage, something that has been more appropriate in vivo until recently.

Validation of the 3D reconstructed human skin Comet assay, an animal-free alternative for following-up positive results from standard in vitro genotoxicity assays.

As part of the safety assessment process, all industrial sectors employ genotoxicity test batteries, starting with well-established in vitro assays. However, these batteries have limited predictive capacity for the in vivo situation, which may result in unnecessary follow-up in vivo testing or the loss of promising substances where animal tests are prohibited or not desired. To address this, a project involving regulators, academia and industry was established to develop and validate in vitro human skin-based genotoxicity assays for topically exposed substances, such as cosmetics ingredients. Here, we describe the validation of the 3D reconstructed skin (RS) Comet assay. In this multicenter study, chemicals were applied topically three times to the skin over 48 h. Isolated keratinocytes and fibroblasts were transferred to slides before electrophoresis and the resulting comet formation was recorded as % tail DNA. Before decoding, results of the validation exercise for 32 substances were evaluated by an independent statistician. There was a high predictive capacity of this assay when compared to in vivo outcomes, with a sensitivity of 77 (80)%, a specificity of 88 (97)% and an overall accuracy of 83 (92)%. The numbers reflect the calls of the performing laboratories in the coded phase, whereas those in parenthesis reflect calls according to the agreed evaluation criteria. Intra- and inter-laboratory reproducibility was also very good, with a concordance of 93 and 88%, respectively. These results generated with the Phenion® Full-Thickness skin model demonstrate its suitability for this assay, with reproducibly low background DNA damage and sufficient metabolic capacity to activate pro-mutagens. The validation outcome supports the use of the RS Comet assay to follow up positive results from standard in vitro genotoxicity assays when the expected route of exposure is dermal. Based on the available data, the assay was accepted recently into the OECD test guideline development program.

A review to support the derivation of a worst-case dermal penetration value for nanoparticles.

Data on dermal penetration of nanoparticles (NPs) was reviewed with the goal to establish a worst-case dermal penetration value for NPs. To this aim, the main focus was on studies providing quantitative dermal penetration data (29 studies). In vivo dermal penetration studies and ex vivo studies based on skin explants were included. These studies used NPs with different compositions, dimensions, and shapes. The overall results showed that skin is an efficient barrier for NPs, indistinctly of their properties. However, some studies reported that a small percentage of the applied NP dose penetrated the skin surface and reached deeper skin layers. The integrity of the skin layer and the product formulation were more critical determinants of dermal penetration than the NP properties. Most quantitative studies were based on elemental analysis such that it cannot be concluded if detected levels are attributable to a dissolved fraction or to the penetration of particles as such. Results of qualitative imaging studies suggest that at least a fraction of the levels reported in quantitative studies could be due to particle penetration. Altogether, based on the data compiled, we propose that 1% could be used as a worst-case dermal penetration value for nanoparticles within the boundaries of the properties of those included in our analysis.

Thermodynamic Parameters at Bio-Nano Interface and Nanomaterial Toxicity: A Case Study on BSA Interaction with ZnO, SiO2, and TiO2.

Understanding nanomaterial (NM)-protein interactions is a key issue in defining the bioreactivity of NMs with great impact for nanosafety. In the present work, the complex phenomena occurring at the bio/nano interface were evaluated in a simple case study focusing on NM-protein binding thermodynamics and protein stability for three representative metal oxide NMs, namely, zinc oxide (ZnO; NM-110), titanium dioxide (TiO2; NM-101), and silica (SiO2; NM-203). The thermodynamic signature associated with the NM interaction with an abundant protein occurring in most cell culture media, bovine serum albumin (BSA), has been investigated by isothermal titration and differential scanning calorimetry. Circular dichroism spectroscopy offers additional information concerning adsorption-induced protein conformational changes. The BSA adsorption onto NMs is enthalpy-controlled, with the enthalpic character (favorable interaction) decreasing as follows: ZnO (NM-110) > SiO2 (NM-203) > TiO2 (NM-101). The binding of BSA is spontaneous, as revealed by the negative free energy, ΔG, for all systems. The structural stability of the protein decreased as follows: TiO2 (NM-101) > SiO2 (NM-203) > ZnO (NM-110). As protein binding may alter NM reactivity and thus the toxicity, we furthermore assessed its putative influence on DNA damage, as well as on the expression of target genes for cell death (RIPK1, FAS) and oxidative stress (SOD1, SOD2, CAT, GSTK1) in the A549 human alveolar basal epithelial cell line. The enthalpic component of the BSA-NM interaction, corroborated with BSA structural stability, matched the ranking for the biological alterations, i.e., DNA strand breaks, oxidized DNA lesions, cell-death, and antioxidant gene expression in A549 cells. The relative and total content of BSA in the protein corona was determined using mass-spectrometry-based proteomics. For the present case study, the thermodynamic parameters at bio/nano interface emerge as key descriptors for the dominant contributions determining the adsorption processes and NMs toxicological effect.

Mass Cytometry Enabling Absolute and Fast Quantification of Silver Nanoparticle Uptake at the Single Cell Level.

In the last decades, significant efforts have been made to investigate possible cytotoxic effects of metallic nanoparticles (NPs). Methodologies enabling precise information regarding uptake and intracellular distribution of NPs at the single cell level remain to be established. Mass cytometry (MC) has been developed for high-dimensional single cell analyses and is a promising tool to quantify NP-cell interactions. Here, we aim to establish a new MC-based quantification procedure to receive absolute numbers of NPs per single cell by using a calibration that considers the specific transmission efficiency (TE) of suspended NPs. The current MC-quantification strategy accept TE values of complementary metal solutions. In this study, we demonstrate the different transmission behavior of 50 nm silver NPs (AgNP) and silver nitrate solution. We have used identical AgNPs for calibration as for in vitro-differentiated macrophages (THP-1 cell line) in a time- and dose-dependent manner. Our quantification relies on silver intensities measuring AgNPs in the same detection mode as the cells. Results were comparable with the TE quantification strategy using AgNPs but differed when using ionic silver. Furthermore, intact and digested cell aliquots were measured to investigate the impact of MC sample processing on the amount of AgNPs/cell. Taken together, we have provided a MC-specific calibration procedure to precisely calculate absolute numbers of NPs per single cell. Combined with its unique feature of multiplexing up to 50 parameters, MC provides much more information on the single cell level than single cell-inductively coupled plasma mass spectrometry (SC-ICPMS) and, therefore, offers new opportunities in nanotoxicology.

An in-depth multi-omics analysis in RLE-6TN rat alveolar epithelial cells allows for nanomaterial categorization.

BACKGROUND: Nanomaterials (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). RESULTS: Cluster 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. CONCLUSIONS: Our 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.

A multi-omics analysis reveals metabolic reprogramming in THP-1 cells upon treatment with the contact allergen DNCB.

Dendritic cell (DC) activation by contact allergens is one of the key steps in the development of allergic contact dermatitis (ACD). Recent evidence suggests that metabolic reprogramming is a prerequisite for the activation of DCs, macrophages and monocytes. Therefore, we used an integrated approach by combining proteomics and metabolomics to investigate the metabolism of human THP-1 cells in response to the strong contact allergen, 2,4-dinitrochlorobenzene (DNCB). Cells were treated with 5, 10 and 20µM DNCB for 4, 8, and 24h, respectively. Using a targeted metabolomics approach, we quantified levels of 188 endogenous metabolites, among them phospholipids, acylcarnitines, amino acids and hexoses. In addition, proteomic changes were analyzed using an untargeted quantitative approach based on stable isotope labeling with amino acids in cell culture (SILAC). We detected several alterations in the metabolome and consistently in the proteome indicating metabolic reprogramming of THP-1 cells by DNCB. In particular, we found an increase in phospholipids that was accompanied by an up-regulation of fatty acid synthase (FAS), a key enzyme in lipid synthesis.

Nanomaterials: certain aspects of application, risk assessment and risk communication.

Development and market introduction of new nanomaterials trigger the need for an adequate risk assessment of such products alongside suitable risk communication measures. Current application of classical and new nanomaterials is analyzed in context of regulatory requirements and standardization for chemicals, food and consumer products. The challenges of nanomaterial characterization as the main bottleneck of risk assessment and regulation are presented. In some areas, e.g., quantification of nanomaterials within complex matrices, the establishment and adaptation of analytical techniques such as laser ablation inductively coupled plasma mass spectrometry and others are potentially suited to meet the requirements. As an example, we here provide an approach for the reliable characterization of human exposure to nanomaterials resulting from food packaging. Furthermore, results of nanomaterial toxicity and ecotoxicity testing are discussed, with concluding key criteria such as solubility and fiber rigidity as important parameters to be considered in material development and regulation. Although an analysis of the public opinion has revealed a distinguished rating depending on the particular field of application, a rather positive perception of nanotechnology could be ascertained for the German public in general. An improvement of material characterization in both toxicological testing as well as end-product control was concluded as being the main obstacle to ensure not only safe use of materials, but also wide acceptance of this and any novel technology in the general public.

Influence of Label and Charge Density on the Association of the Therapeutic Monoclonal Antibodies Trastuzumab and Cetuximab Conjugated to Anionic Fluorophores.

The design of bright and functional dye-protein conjugates requires hydrophilic and stable fluorophores with high molar absorption coefficients and high fluorescence quantum yields, which must not be prone to dimerization, as well as conservation of protein function and suppression of protein association. Although many synthetic dyes meet these needs, the influence of dye charge on bioconjugate performance is commonly neglected. This encouraged us to assess the spectroscopic properties, antibody functionality, binding behavior, folding, and association of conjugates of the therapeutic antibodies trastuzumab and cetuximab with the red cyanine dyes S0586, S2381, and 6SIDCC (bearing two, three, and six sulfonate groups, respectively). Our results demonstrate a negligible effect of dye labeling on antibody folding, yet a strong influence of label charge and density on antibody isoelectric points and association. Especially 6SIDCC decreased strongly the isoelectric points of both antibodies and their heavy or light chains even at low labeling degrees, thus favoring protein association. Although an increasingly negative dye charge reduces antigen affinity as shown in a competitive immunoassay, all conjugates still bound to cells overexpressing the target of the respective antibody. Obviously, dyes that cause minimum dimerization with a small number of charged groups are best for conjugate brightness, minimum protein association, and strong target binding. This underlines the need to consider dye charge for the rational design of conjugates with optimum performance.

Quantification of silver nanoparticle uptake and distribution within individual human macrophages by FIB/SEM slice and view.

BACKGROUND: Quantification of nanoparticle (NP) uptake in cells or tissues is very important for safety assessment. Often, electron microscopy based approaches are used for this purpose, which allow imaging at very high resolution. However, precise quantification of NP numbers in cells and tissues remains challenging. The aim of this study was to present a novel approach, that combines precise quantification of NPs in individual cells together with high resolution imaging of their intracellular distribution based on focused ion beam/ scanning electron microscopy (FIB/SEM) slice and view approaches. RESULTS: We quantified cellular uptake of 75 nm diameter citrate stabilized silver NPs (Ag 75 Cit) into an individual human macrophage derived from monocytic THP-1 cells using a FIB/SEM slice and view approach. Cells were treated with 10 µg/ml for 24 h. We investigated a single cell and found in total 3138 ± 722 silver NPs inside this cell. Most of the silver NPs were located in large agglomerates, only a few were found in clusters of fewer than five NPs. Furthermore, we cross-checked our results by using inductively coupled plasma mass spectrometry and could confirm the FIB/SEM results. CONCLUSIONS: Our approach based on FIB/SEM slice and view is currently the only one that allows the quantification of the absolute dose of silver NPs in individual cells and at the same time to assess their intracellular distribution at high resolution. We therefore propose to use FIB/SEM slice and view to systematically analyse the cellular uptake of various NPs as a function of size, concentration and incubation time.

Genotoxicity testing of different surface-functionalized SiO2, ZrO2 and silver nanomaterials in 3D human bronchial models.

Inhalation is considered a critical uptake route for NMs, demanding for sound toxicity testing using relevant test systems. This study investigates cytotoxicity and genotoxicity in EpiAirway™ 3D human bronchial models using 16 well-characterized NMs, including surface-functionalized 15 nm SiO2 (4 variants), 10 nm ZrO2 (4), and nanosilver (3), ZnO NM-110, TiO2 NM-105, BaSO4 NM-220, and two AlOOH NMs. Cytotoxicity was assessed by LDH and ATP assays and genotoxicity by the alkaline comet assay. For 9 NMs, uptake was investigated using inductively coupled plasma-mass spectrometry (ICP-MS). Most NMs were neither cytotoxic nor genotoxic in vitro. ZnO displayed a dose-dependent genotoxicity between 10 and 25 µg/cm2. Ag.50.citrate was genotoxic at 50 µg/cm2. A marginal but still significant genotoxic response was observed for SiO2.unmodified, SiO2.phosphate and ZrO2.TODS at 50 µg/cm2. For all NMs for which uptake in the 3D models could be assessed, the amount taken up was below 5% of the applied mass doses and was furthermore dose dependent. For in vivo comparison, published in vivo genotoxicity data were used and in addition, at the beginning of this study, two NMs were randomly selected for short-term (5-day) rat inhalation studies with subsequent comet and micronucleus assays in lung and bone marrow cells, respectively, i.e., ZrO2.acrylate and SiO2.amino. Both substances were not genotoxic neither in vivo nor in vitro. EpiAirway™ 3D models appear useful for NM in vitro testing. Using 16 different NMs, this study confirms that genotoxicity is mainly determined by chemical composition of the core material.

Pan-European inter-laboratory studies on a panel of in vitro cytotoxicity and pro-inflammation assays for nanoparticles.

The rapid development of nanotechnologies and increased production and use of nanomaterials raise concerns about their potential toxic effects for human health and environment. To evaluate the biological effects of nanomaterials, a set of reliable and reproducible methods and development of standard operating procedures (SOPs) is required. In the framework of the European FP7 NanoValid project, three different cell viability assays (MTS, ATP content, and caspase-3/7 activity) with different readouts (absorbance, luminescence and fluorescence) and two immune assays (ELISA of pro-inflammatory cytokines IL1-ß and TNF-α) were evaluated by inter-laboratory comparison. The aim was to determine the suitability and reliability of these assays for nanosafety assessment. Studies on silver and copper oxide nanoparticles (NPs) were performed, and SOPs for particle handling, cell culture, and in vitro assays were established or adapted. These SOPs give precise descriptions of assay procedures, cell culture/seeding conditions, NPs/positive control preparation and dilutions, experimental well plate preparation, and evaluation of NPs interference. The following conclusions can be highlighted from the pan-European inter-laboratory studies: Testing of NPs interference with the toxicity assays should always be conducted. Interference tests should be designed as close as possible to the cell exposure conditions. ATP and MTS assays gave consistent toxicity results with low inter-laboratory variability using Ag and CuO NPs and different cell lines and therefore, could be recommended for further validation and standardization. High inter-laboratory variability was observed for Caspase 3/7 assay and ELISA for IL1-ß and TNF-α measurements.

Systems Biology to Support Nanomaterial Grouping.

The assessment of potential health risks of engineered nanomaterials (ENMs) is a challenging task due to the high number and great variety of already existing and newly emerging ENMs. Reliable grouping or categorization of ENMs with respect to hazards could help to facilitate prioritization and decision making for regulatory purposes. The development of grouping criteria, however, requires a broad and comprehensive data basis. A promising platform addressing this challenge is the systems biology approach. The different areas of systems biology, most prominently transcriptomics, proteomics and metabolomics, each of which provide a wealth of data that can be used to reveal novel biomarkers and biological pathways involved in the mode-of-action of ENMs. Combining such data with classical toxicological data would enable a more comprehensive understanding and hence might lead to more powerful and reliable prediction models. Physico-chemical data provide crucial information on the ENMs and need to be integrated, too. Overall statistical analysis should reveal robust grouping and categorization criteria and may ultimately help to identify meaningful biomarkers and biological pathways that sufficiently characterize the corresponding ENM subgroups. This chapter aims to give an overview on the different systems biology technologies and their current applications in the field of nanotoxicology, as well as to identify the existing challenges.

A redox proteomics approach to investigate the mode of action of nanomaterials.

Numbers of engineered nanomaterials (ENMs) are steadily increasing. Therefore, alternative testing approaches with reduced costs and high predictivity suitable for high throughput screening and prioritization are urgently needed to ensure a fast and effective development of safe products. In parallel, extensive research efforts are targeted to understanding modes of action of ENMs, which may also support the development of new predictive assays. Oxidative stress is a widely accepted paradigm associated with different adverse outcomes of ENMs. It has frequently been identified in in vitro and in vivo studies and different assays have been developed for this purpose. Fluorescent dye based read-outs are most frequently used for cell testing in vitro but may be limited due to possible interference of the ENMs. Recently, other assays have been put forward such as acellular determination of ROS production potential using methods like electron spin resonance, antioxidant quantification or the use of specific sensors. In addition, Omics based approaches have gained increasing attention. In particular, redox proteomics can combine the assessment of oxidative stress with the advantage of getting more detailed mechanistic information. Here we propose a comprehensive testing strategy for assessing the oxidative stress potential of ENMs, which combines acellular methods and fast in vitro screening approaches, as well as a more involved detailed redox proteomics approach. This allows for screening and prioritization in a first tier and, if required, also for unraveling mechanistic details down to compromised signaling pathways.

Proteomics analysis of dendritic cell activation by contact allergens reveals possible biomarkers regulated by Nrf2.

Allergic contact dermatitis is a widespread disease with high clinical relevance affecting approximately 20% of the general population. Typically, contact allergens are low molecular weight electrophilic compounds which can activate the Keap1/Nrf2 pathway. We performed a proteomics study to reveal possible biomarkers for dendritic cell (DC) activation by contact allergens and to further elucidate the role of Keap1/Nrf2 signaling in this process. We used bone marrow derived dendritic cells (BMDCs) of wild-type (nrf2+/+) and Nrf2 knockout (nrf2-/-) mice and studied their response against the model contact sensitizers 2,4-dinitrochlorobenzene (DNCB), cinnamaldehyde (CA) and nickel(II) sulfate by 2-dimensional polyacrylamide gel electrophoresis (2D-PAGE) in combination with electrospray ionization tandem mass spectrometry (ESI-MS/MS). Sodium dodecyl sulfate (SDS, 100µM) served as irritant control. While treatment with nickel(II) sulfate and SDS had only little effects, CA and DNCB led to significant changes in protein expression. We found 18 and 30 protein spots up-regulated in wild-type cells treated with 50 and 100µM CA, respectively. For 5 and 10µM DNCB, 32 and 37 spots were up-regulated, respectively. Almost all of these proteins were not differentially expressed in nrf2-/- BMDCs, indicating an Nrf2-dependent regulation. Among them proteins were detected which are involved in oxidative stress and heat shock responses, as well as in signal transduction or basic cellular pathways. The applied approach allowed us to differentiate between Nrf2-dependent and Nrf2-independent cellular biomarkers differentially regulated upon allergen-induced DC activation. The data presented might contribute to the further development of suitable in vitro testing methods for chemical-mediated sensitization.

Quantification and visualization of cellular uptake of TiO2 and Ag nanoparticles: comparison of different ICP-MS techniques.

BACKGROUND: Safety assessment of nanoparticles (NPs) requires techniques that are suitable to quantify tissue and cellular uptake of NPs. The most commonly applied techniques for this purpose are based on inductively coupled plasma mass spectrometry (ICP-MS). Here we apply and compare three different ICP-MS methods to investigate the cellular uptake of TiO2 (diameter 7 or 20 nm, respectively) and Ag (diameter 50 or 75 nm, respectively) NPs into differentiated mouse neuroblastoma cells (Neuro-2a cells). Cells were incubated with different amounts of the NPs. Thereafter they were either directly analyzed by laser ablation ICP-MS (LA-ICP-MS) or were lysed and lysates were analyzed by ICP-MS and by single particle ICP-MS (SP-ICP-MS). RESULTS: All techniques confirmed that smaller particles were taken up to a higher extent when values were converted in an NP number-based dose metric. In contrast to ICP-MS and LA-ICP-MS, this measure is already directly provided through SP-ICP-MS. Analysis of NP size distribution in cell lysates by SP-ICP-MS indicates the formation of NP agglomerates inside cells. LA-ICP-MS imaging shows that some of the 75 nm Ag NPs seemed to be adsorbed onto the cell membranes and were not penetrating into the cells, while most of the 50 nm Ag NPs were internalized. LA-ICP-MS confirms high cell-to-cell variability for NP uptake. CONCLUSIONS: Based on our data we propose to combine different ICP-MS techniques in order to reliably determine the average NP mass and number concentrations, NP sizes and size distribution patterns as well as cell-to-cell variations in NP uptake and intracellular localization.

Proteomic analysis of protein carbonylation: a useful tool to unravel nanoparticle toxicity mechanisms.

BACKGROUND: Oxidative 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. METHODS: We 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. RESULTS: Eleven 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. CONCLUSIONS: Analysis 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.