Differentially\nInduced Autophagy by Engineered Nanomaterial\nTreatment Has an Impact on Cellular Homeostasis and Cytotoxicity

Considering the increasing production of engineered nanomaterials\n(ENMs), new approach methodologies (NAMs) are essential for safe-by-design\napproaches and risk assessment. Our aim was to enhance screening strategies\nwith a focus on reactivity-triggered toxicities. We applied <i>in vitro</i> tests to 10 selected benchmark ENMs in two cell\nmodels, lung epithelial A549 and differentiated THP-1 macrophage-like\ncells. Previously, we categorized ENMs based on surface reactivity.\nHere we elucidated their reactivity-triggered cytotoxicity and mode\nof action using the WST-1 assay (metabolic activity), LDH assay (cell\nmembrane integrity), autophagosome detection, and proteomics. Nonreactive\nSiO<sub>2</sub> NM-200 showed no significant impact on cell viability.\nConversely, highly reactive CuO and ZnO (NM-110 and NM-111) disrupted\ncell homeostasis. Interestingly, moderately reactive TiO<sub>2</sub> (NM-101 and NM-105) and CeO<sub>2</sub> (NM-211 and NM-212), apparently\nwithout an adverse effect, induced autophagosome formation, evidencing\nautophagy as a defensive mechanism. Our improved <i>in vitro</i> testing strategy, combined with state-of-the-art reactivity information,\nscreens ENMs for potential reactivity-triggered toxicity.