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.

Advancing Nanomaterial Toxicology Screening Through Efficient and Cost-Effective Quantitative Proteomics.

Proteomic investigations yield high-dimensional datasets, yet their application to large-scale toxicological assessments is hindered by reproducibility challenges due to fluctuating measurement conditions. To address these limitations, this study introduces an advanced tandem mass tag (TMT) labeling protocol. Although labeling approaches shorten data acquisition time by multiplexing samples compared to traditional label-free quantification (LFQ) methods in general, the associated costs may surge significantly with large sample sets, for example, in toxicological screenings. However, the introduced advanced protocol offers an efficient, cost-effective alternative, reducing TMT reagent usage (by a factor of ten) and requiring minimal biological material (1 µg), while demonstrating increased reproducibility compared to LFQ. To demonstrate its effectiveness, the advanced protocol is employed to assess the toxicity of nine benchmark nanomaterials (NMs) on A549 lung epithelial cells. While LFQ measurements identify 3300 proteins, they proved inadequate to reveal NM toxicity. Conversely, despite detecting 2600 proteins, the TMT protocol demonstrates superior sensitivity by uncovering alterations induced by NM treatment. In contrast to previous studies, the introduced advanced protocol allows simultaneous and straightforward assessment of multiple test substances, enabling prioritization, ranking, and grouping for hazard evaluation. Additionally, it fosters the development of New Approach Methodologies (NAMs), contributing to innovative methodologies in toxicological research.