Comprehensive In Vitro Toxicity Testing of a Panel of Representative Oxide Nanomaterials: First Steps towards an Intelligent Testing Strategy

Nanomaterials (NMs) display many unique and useful physico-chemical properties. However, reliable approaches are needed for risk assessment of NMs. The present study was performed in the FP7-MARINA project, with the objective to identify and evaluate in vitro test methods for toxicity assessment in order to facilitate the development of an intelligent testing strategy (ITS). Six representative oxide NMs provided by the EC-JRC Nanomaterials Repository were tested in nine laboratories. The in vitro toxicity of NMs was evaluated in 12 cellular models representing 6 different target organs/systems (immune system, respiratory system, gastrointestinal system, reproductive organs, kidney and embryonic tissues). The toxicity assessment was conducted using 10 different assays for cytotoxicity, embryotoxicity, epithelial integrity, cytokine secretion and oxidative stress. Thorough physico-chemical characterization was performed for all tested NMs. Commercially relevant NMs with different physico-chemical properties were selected: two TiO2 NMs with different surface chemistry – hydrophilic (NM-103) and hydrophobic (NM-104), two forms of ZnO – uncoated (NM-110) and coated with triethoxycapryl silane (NM-111) and two SiO2 NMs produced by two different manufacturing techniques – precipitated (NM-200) and pyrogenic (NM-203). Cell specific toxicity effects of all NMs were observed; macrophages were the most sensitive cell type after short-term exposures (24-72h) (ZnO>SiO2>TiO2). Longer term exposure (7 to 21 days) significantly affected the cell barrier integrity in the presence of ZnO, but not TiO2 and SiO2, while the embryonic stem cell test (EST) classified the TiO2 NMs as potentially ‘weak-embryotoxic’ and ZnO and SiO2 NMs as ‘non-embryotoxic’. A hazard ranking could be established for the representative NMs tested (ZnO NM-110 > ZnO NM-111 > SiO2 NM-203 > SiO2 NM-200 > TiO2 NM-104 > TiO2 NM-103). This ranking was different in the case of embryonic tissues, for which TiO2 displayed higher toxicity compared with ZnO and SiO2. Importantly, the in vitro methodology applied could identify cell- and NM-specific responses, with a low variability observed between different test assays. Overall, this testing approach, based on a battery of cellular systems and test assays, complemented by an exhaustive physico-chemical characterization of NMs, could be deployed for the development of an ITS suitable for risk assessment of NMs. This study also provides a rich source of data for modeling of NM effects

A multi-country test of brief reappraisal interventions on emotions during the COVID-19 pandemic.

The COVID-19 pandemic has increased negative emotions and decreased positive emotions globally. Left unchecked, these emotional changes might have a wide array of adverse impacts. To reduce negative emotions and increase positive emotions, we tested the effectiveness of reappraisal, an emotion-regulation strategy that modifies how one thinks about a situation. Participants from 87 countries and regions (n = 21,644) were randomly assigned to one of two brief reappraisal interventions (reconstrual or repurposing) or one of two control conditions (active or passive). Results revealed that both reappraisal interventions (vesus both control conditions) consistently reduced negative emotions and increased positive emotions across different measures. Reconstrual and repurposing interventions had similar effects. Importantly, planned exploratory analyses indicated that reappraisal interventions did not reduce intentions to practice preventive health behaviours. The findings demonstrate the viability of creating scalable, low-cost interventions for use around the world

“Uh” and “um” in autism: The case of hesitation marker usage in Dutch-speaking autistic preschoolers

English-speaking autistic children use the hesitation marker “um” less often than non-autistic children while they use “uh” at a similar rate. It is unclear why this is the case. We examined hesitation marker usage of Dutch-speaking autistic and non-autistic preschoolers with the aim to 1) make a crosslinguistic comparison of hesitation marker usage and 2) examine two hypotheses regarding the underlying linguistic mechanisms of hesitation markers: the symptom hypothesis and the signal hypothesis. We replicated group differences in “um” usage and showed significant effects of age and biological sex in the selection of hesitation marker (“uh” versus “um”). We found that “uh” usage is correlated with structural language abilities while “um” is not. “Um” may be related to pragmatic language abilities, which were not examined here. The findings support the notion that hesitation markers are not involuntary utterances but deliberate language features, which is in line with the signal hypothesis

A cross-linguistic examination of language measures in autism: A comparison between Dutch and English

Background: Language abilities are highly heterogeneous in autism. While a multimodal assessment approach is recommended to capture the language variability, this is not always possible. Therefore, it is important to gain contextual information about different language assessments to determine which assessment is most appropriate for different research questions. As most current work is based on English-speaking populations, this paper compares three language assessment modalities (standardized assessment, parent survey, and a natural language sample) between English-speaking and Dutch-speaking autistic and neurotypical children. Method: The Mullen Scales of Early Learning, the Vineland Adaptive Behavior Scales and a naturalistic language sample were employed to measure language in 100 preschool-aged participants. Correlation analyses and mixed-effect regressions were conducted and Bland-Altman plots were created to examine the similarity between measures. Results: English-speaking and Dutch-speaking parents rated their children’s expressive language higher than their receptive language. The best agreement for between measures was for standardized language and parent-report. Agreement was higher for children with low language scores. Primary language (English vs. Dutch) did not significantly affect the results. Conclusions: For autistic children with low language levels, parent-reported and standardized language measures provide researchers with similar information. Depending on the available time and resources, researchers may choose to use one of these methods. For autistic children with (above) average language abilities however, multiple modalities should be considered to gain a comprehensive understanding of their language abilities across different settings. A natural language sample is of most added value next to a standardized assessment or parent report

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

Additional file 1: Figure S1-S5 of Proteomic analysis of protein carbonylation: a useful tool to unravel nanoparticle toxicity mechanisms

and Table S1. The file contains various additional figures and tables, collected in a single portable documend file (PDF) (DOC 9935 kb)

Influence of agglomeration and specific lung lining lipid/protein interaction on short-term inhalation toxicity

<p>Lung lining fluid is the first biological barrier nanoparticles (NPs) encounter during inhalation. As previous inhalation studies revealed considerable differences between surface functionalized NPs with respect to deposition and toxicity, our aim was to investigate the influence of lipid and/or protein binding on these processes. Thus, we analyzed a set of surface functionalized NPs including different SiO₂ and ZrO₂ in pure phospholipids, CuroSurf^TM and purified native porcine pulmonary surfactant (nS). Lipid binding was surprisingly low for pure phospholipids and only few NPs attracted a minimal lipid corona. Additional presence of hydrophobic surfactant protein (SP) B in CuroSurf^TM promoted lipid binding to NPs functionalized with Amino or PEG residues. The presence of the hydrophilic SP A in nS facilitated lipid binding to all NPs. In line with this the degree of lipid and protein affinities for different surface functionalized SiO₂ NPs in nS followed the same order (SiO₂ Phosphate ∼ unmodified SiO₂ < SiO₂ PEG < SiO₂ Amino NPs). Agglomeration and biomolecule interaction of NPs in nS was mainly influenced by surface charge and hydrophobicity. Toxicological differences as observed in short-term inhalation studies (STIS) were mainly influenced by the core composition and/or surface reactivity of NPs. However, agglomeration in lipid media and lipid/protein affinity appeared to play a modulatory role on short-term inhalation toxicity. For instance, lipophilic NPs like ZrO₂, which are interacting with nS to a higher extent, exhibited a far higher lung burden than their hydrophilic counterparts, which deserves further attention to predict or model effects of respirable NPs.</p