Cytoprotective Mechanism of Necrox-5 Against Toxicity Induced by Experimental Ferroptosis Instigators and the Pesticide Propargite

Necrox-5 is an indole-derived antioxidant that inhibits necrotic cell death, likely through prevention of mitochondrial stress, oxidative stress, inflammation, and hypoxia/reoxygenation. However, its protective role against ferroptotic toxicity has not yet been studied. In this study, we induced ferroptosis in HT-22 cells, an immortalized hippocampal neuronal cell line, using ferroptosis-inducing agents. We also tested Necrox-5 against toxicity induced by propargite, a pesticide known to inhibit complex V (mitochondrial adenosine triphosphate [ATP] synthase) and induce necrosis. We evaluated cytotoxicity using calcein AM and lactate dehydrogenase (LDH) release assays. Additionally, we conducted intracellular and cell-free C11-BODIPY assays to assess the efficacy of Necrox-5 in inhibiting lipid peroxidation. Intracellular glutathione (GSH) levels were measured using the mBCI-GSH assay, while ATP levels were determined through bioluminescence assays. Our findings show that Necrox-5 is a potent inhibitor of ferroptosis induced by erastin, RSL3, FINO2, and iron plus arachidonic acid. Furthermore, we demonstrated that Necrox-5 protects against ferroptosis-like propargite toxicity, although it did not prevent propargite-induced depletion of GSH and ATP. We identified radical-scavenging antioxidant activity as the primary mechanism by which Necrox-5 protects from ferroptosis and propargite toxicity. In conclusion, Necrox-5 is a potent cytoprotective compound that warrants further study for its potential role in ferroptosis-associated complications such as neurodegenerative diseases.