ALDH1A3-dependent ferroptosis mediates hexafluoropropylene oxide dimer acid-induced trophoblast dysfunction

Hexafluoropropylene oxide dimer acid (HFPO-DA), a persistent per- and polyfluoroalkyl substance, has been epidemiologically associated with adverse pregnancy outcomes; however, the molecular mechanisms underlying its placental toxicity remain poorly defined. Here, we demonstrate that acute 24-h exposure of human placental trophoblast HTR-8/SVneo and JEG-3 cells to HFPO-DA (0.03-3 μM) induces a concentration-dependent impairment in cell viability, proliferation, migration, and invasion, whereas inhibitors of apoptosis, necroptosis, and pyroptosis did not show comparable protective effects. Integrated bioinformatic analysis of HFPO-DA-associated pregnancy complication genes retrieved from the Comparative Toxicogenomics Database revealed significant enrichment of ferroptosis-related pathways. Protein-protein interaction network analysis further identified ALDH1A3 as a central hub gene, and molecular docking predicted a strong binding affinity between HFPO-DA and ALDH1A3, exceeding that observed for other candidate targets. Biolayer interferometry further confirmed a direct interaction between HFPO-DA and ALDH1A3, with an apparent equilibrium dissociation constant (KD) of 6.64 × 10^-4 M. Consistent with these in silico findings, pharmacological inhibition experiments showed that only the ferroptosis inhibitor ferrostatin-1 effectively rescued HFPO-DA-induced cytotoxicity. HFPO-DA exposure led to hallmark ferroptotic alterations, including glutathione and superoxide dismutase depletion, malondialdehyde accumulation, intracellular Fe²⁺ overload, GPX4 suppression, and increased mitochondrial superoxide production in both trophoblast cell models. Mechanistically, HFPO-DA markedly downregulated ALDH1A3 at both mRNA and protein levels. Notably, siRNA-mediated ALDH1A3 silencing alone recapitulated ferroptosis-associated biochemical and functional defects, whereas ALDH1A3 overexpression restored redox homeostasis, attenuated lipid peroxidation and iron dysregulation, and rescued trophoblast functional impairment. Collectively, these findings identify ALDH1A3 repression as a key molecular event linking HFPO-DA exposure to trophoblast ferroptosis and dysfunction, providing mechanistic insight into HFPO-DA-associated placental pathogenesis.