Hexafluoropropylene oxide dimer acid (GenX) induces apoptosis in primary cortical neurons via stimulating ROS production and NF-κB activation.

Hexafluoropropylene oxide dimer acid (HFPO-DA), commonly known as GenX, is a replacement for perfluorooctanoic acid (PFOA) which readily accumulates in the brain and exhibits neurotoxic effects. However, the adverse impacts of GenX on neurons and its underlying mechanisms remain poorly understood. In this study, primary cortical neurons isolated from neonatal mice were exposed to varying concentrations of GenX to assess cell viability, intracellular reactive oxygen species (ROS) levels, and morphological alterations. Additionally, the expression of apoptosis-related proteins Bcl-2, Bax, Caspase-3, NF-κB, and Tomm20 was examined. The results showed that increasing concentrations of GenX significantly elevated intracellular ROS levels and markedly reduced cell viability and the number of cells. Neuronal morphology was severely disrupted, characterized by decreased neurite branching, shortened neurite length, and reduced soma size. At 200 μM and 400 μM GenX, apoptosis rates were dramatically increased (p < 0.0001), accompanied by a pronounced increase in NF-κB fluorescence intensity and nuclear translocation. Western blot analysis further revealed a progressive downregulation of Bcl-2 and Tomm20, while levels of Bax, Cleaved Caspase-3/Caspase-3 increased in a dose-dependent manner. Notably, pretreatment with N-Acetylcysteine (NAC) effectively reversed GenX-induced ROS accumulation (p = 0.0001), NF-κB activation, and neuronal apoptosis. Collectively, these findings demonstrate that GenX exposure induces ROS accumulation in primary cortical neurons, leading to apoptosis through mitochondrial dysfunction mediated by Tomm20 downregulation and the activation of Caspase-3 and NF-κB. This study provides novel mechanistic insights into the neurotoxicity of the emerging environmental contaminant GenX and offers a theoretical basis for developing neuroprotective targets against such exposures.