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 <i>in vitro</i> 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 <i>in vitro</i> testing strategy, combined with state-of-the-art reactivity information, screens ENMs for potential reactivity-triggered toxicity.