Ferritinophagy-derived iron causes protein nitration and mitochondrial dysfunction in acetaminophen-induced liver injury.

Acetaminophen (APAP), also known as paracetamol, is a widely used analgesic and antipyretic drug. While the drug is effective and safe at recommended doses, excessive intake can lead to acute liver injury (ALI) due to the formation of the toxic metabolite N-acetyl-p-benzoquinone imine (NAPQI), which depletes glutathione (GSH). Despite regulatory efforts, APAP-related liver injury remains a significant health concern. However, the cellular pathways that contribute to APAP-induced hepatotoxicity-particularly those involving iron metabolism-remain incompletely understood. To address this gap, we investigated whether ferritinophagy-the autophagic degradation of ferritin heavy chain (FTH) mediated by nuclear receptor coactivator 4 (NCOA4)-contributes to APAP-induced ALI. We administered APAP to C57BL/6 J mice and AML-12 hepatocyte cells and monitored markers of ferritinophagy, iron release, and hepatic injury. In parallel, we assessed the protective effect of the iron chelator deferoxamine (DFO) to validate the pathogenic role of free iron in vivo. First, in vivo studies revealed that APAP treatment significantly upregulated NCOA4 and FTH mRNA expression at 6 h post-exposure, coupled with increased LC3II protein and decreased p62, NCOA4, and FTH protein levels-hallmarks of active ferritinophagy. Importantly, pretreatment of mice with DFO markedly attenuated serum ALT elevation and histopathological liver damage, indicating that iron released via ferritinophagy critically mediates APAP-induced hepatotoxicity. To corroborate these findings at the cellular level, we measured free iron and ferritinophagy-related proteins in AML-12 cells following APAP exposure. We observed a progressive increase in free iron, with FTH protein level peaking at 2 h and subsequently declining by 6 and 12 h. Concurrently, LC3II protein level rose while NCOA4 protein decreased at 6 h, confirming activation of ferritinophagy in vitro. Although canonical ferroptosis is driven by iron-catalyzed lipid peroxidation (LPO), our APAP model did not exhibit key ferroptotic signatures. In vivo, malondialdehyde (MDA) level and Ptgs2 mRNA did not increase significantly, nor did GPX4 protein level decrease after APAP administration. Similarly, AML-12 cells failed to show a significant rise in C11-BODIPY oxidation after APAP treatment. Thus, APAP-induced ferritinophagy doesn't result in significant LPO. Instead of LPO, APAP exposure led to pronounced protein nitration and mitochondrial dysfunction. Specifically, the protein level of nitrotyrosine (NT) increased significantly at 6 h in vivo, while AML-12 cells exhibited elevated mitochondrial reactive oxygen species (MtROS) alongside reduced mitochondrial membrane potential (MMP) and ATP level. Collectively, these data suggest that ferritinophagy-derived iron triggers protein nitration and mitochondrial impairment, culminating in cell death. Given NCOA4's central role in ferritinophagy, we next evaluated whether its knock-down could mitigate APAP-induced mitochondrial dysfunction. NCOA4 siRNA in AML-12 cells restored ATP level, enhanced MMP, and reduced Fe2+ accumulation and MtROS generation after APAP treatment. Overall, our findings illuminate ferritinophagy-derived iron as a critical driver of APAP hepatotoxicity and nominate NCOA4 inhibition as a promising therapeutic strategy against APAP-induced ALI.