Bioinformatics and experimental insights into the relationship between ferroptosis and fluoride-induced neurotoxicity

Although fluoride is known to cause neurological impairment, the underlying molecular mechanisms remain unclear. Given that ferroptosis may contribute to neurotoxicity, we explored whether fluoride induces neuronal ferroptosis and sought to identify the signaling pathways involved by integrating bioinformatics analysis with experimental validation. Bioinformatics screening of 23 intersecting differentially expressed genes (DEGs) revealed significant enrichment of ferroptosis-related signaling pathways, with TP53 as the top-ranked gene. In silico analysis further predicted three potential p53 binding sites in the SLC7A11 promoter region. Experimentally, rats received 10, 50 and 100 mg/L NaF, and SH-SY5Y cells were treated with 20, 40 and 60 mg/L NaF. NaF-treated rats exhibited impaired performance in Morris water maze tests. Concurrently, elevated Fe^2 + and total iron were detected in NaF-treated rat brains and SH-SY5Y cells. NaF treatment also impaired antioxidant capacity and induced lipid peroxidation, increasing ROS and MDA levels while decreasing GSH levels and the GSH/GSSG ratio, confirmed by immunofluorescence and biochemical analyses in vivo and in vitro. Transmission electron microscopy revealed that NaF induced mitochondrial alterations in vitro, including a loss of cristae. At the molecular level, NaF upregulated p53 (confirmed by immunofluorescence) and ACSL4 while downregulating SLC7A11 (xCT) and GPX4 expression in rat hippocampi and cells. Notably, these pathological alterations were attenuated by the ferroptosis inhibitor Ferrostatin-1 and p53 siRNA, validating the involvement of ferroptosis and the p53/xCT/GPX4 signaling pathway. In conclusion, our findings uncover that fluoride may activate the p53/xCT/GPX4 signaling pathway, inducing neuronal ferroptosis and subsequent neurological impairment, and suggest TP53 as a potential target gene for the control of fluoride neurotoxic effects.