ROS-driven rewiring of Hippo-inflammation-polycomb axis by PFOA in 2D and 3D lung epithelial models

Perfluorooctanoic acid (PFOA), a persistent organic pollutant and prominent member of the per- and polyfluoroalkyl substances (PFAS) family, continues to raise global concern due to its bioaccumulation and potential for chronic human exposure. While hepatic and systemic toxicities of PFOA are well documented, its effects on lung epithelial integrity/homeostasis, particularly at environmentally relevant concentrations, remain incompletely understood. In this study, we investigated the cellular and molecular responses to PFOA in A549 human adenocarcinoma epithelial cells (alveolar type-II like cells) cultured under submerged monolayer (2D) and 3D air-liquid interface (ALI) condition, representing systemic and barrier-relevant exposure models respectively. Cells were exposed to 10-1000 nM PFOA for 24 h, during which we observed robust induction of pro-inflammatory mediators including- transcription factors (NF-κB and STAT3), pattern recognition receptors (TLR4 and RAGE), cytokine/chemokine production (IL-6, IL-8, CCL2, CCL5), and damage-associated molecular patterns (HSP70, HMGB1). To determine whether these inflammatory responses were linked to broader stress‑adaptation and epigenetic reprogramming, we further examined pathways governing post-transcriptional and translational control, mechanistic signaling, and chromatin regulation. PFOA appear to induce translational stress responses as evidenced by increased stress granule and P-body formation. Alterations in Hippo signaling characterized by YAP/TAZ induction and nuclear translocation was also observed in our experimental models. PFOA-challenged cells exhibited enhanced expression of Polycomb Repressive Complex components, along with increased repressive histone marks (H3K27me3, H2AK119Ub). Additionally, concurrent modulation of HDACs and SIRT family members together with these Polycomb-associated changes, suggests stress adaptive epigenetic reprogramming. Oxidative stress emerged as a key upstream mediator of both the epigenetic and Hippo pathway disruptions, as pretreatment with 1 mM N-acetylcysteine (NAC) effectively attenuated these effects. Despite these molecular alterations, epithelial cell migration capacity remains unaffected under acute exposure. Our results provide key mechanistic insight into PFOA-mediated disruption of redox homeostasis, immune/Hippo signaling, and epigenetic plasticity in A549 lung epithelial cells, and identify potential biomarkers to support future biomonitoring efforts and environmental exposure and risk assessment.