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The use of mass spectrometry-based proteomics has been increasingly applied in nanomaterials risk assessments as it provides a proteome-wide overview of the molecular disturbances induced by its exposure. Here, we used this technique to gain detailed molecular insights into the role of ROS as an effector of AgNP toxicity, by incubating Bend3 cells with AgNP in the absence or presence of an antioxidant N-acetyl L-cystein (NAC). ROS generation is a key player in AgNP-induced toxicity, as cellular homeostasis was kept in the presence of NAC. By integrating MS/MS data with bioinformatics tools, in the absence of NAC, we were able to pinpoint precisely which biological pathways were affected by AgNP. Cells respond to AgNP-induced ROS generation by increasing their antioxidant pool, via NRF2 pathway activation. Additionally, cell proliferation-related pathways were strongly inhibited in a ROS-dependent manner. These findings reveal important aspects of the AgNP mechanism of action at the protein level.

Proteome-wide analysis reveals molecular pathways affected by AgNP in a ROS-dependent manner