Toxicological assessment of GenX in WPMY-1 normal prostate stromal cells: apoptotic responses, oxidative stress, and multi-pathway transcriptomic remodeling

Abstract GenX (ammonium perfluoro(2-methyl-3-oxahexanoate)) is an HFPO-DA-related PFAS replacement with emerging health concerns, yet its effects on prostate stromal cells remain poorly understood. This study investigated the cytotoxic, apoptotic, and oxidative effects of GenX in WPMY-1 human prostate stromal cells and characterized the accompanying transcriptomic remodeling. WPMY-1 cells were exposed to 0–50 nM GenX for 24 h; viability was assessed by MTT assay, apoptotic and antioxidant markers (PARP, cleaved PARP, caspase-3, cleaved caspase-3, caspase-9, Bax, Bcl-2, SOD2, catalase) were evaluated by Western blot with Z-VAD-FMK and NAC rescue experiments, and intracellular ROS was measured by DCFDA imaging. Transcriptomic profiling via mRNA-seq (|fold change| ≥ 1.5) was followed by GO, KEGG, and Reactome enrichment analyses, and protein–protein interactions were analyzed using STRING with MCL clustering (inflation parameter = 2.0). GenX induced a dose-dependent viability decrease in WPMY-1 cells at 25 and 50 nM, increased the cleaved PARP/PARP and cleaved caspase-3/caspase-3 ratios, elevated Bax and reduced Bcl-2 at 50 nM, and increased intracellular ROS with concomitant SOD2 and catalase upregulation; Z-VAD-FMK attenuated apoptotic marker cleavage, and NAC attenuated ROS accumulation and antioxidant protein induction. Transcriptomic analysis identified 218 differentially expressed genes (113 upregulated, 105 downregulated), with enriched terms and pathways associated with stress-responsive transcription, MAPK and ER-stress signaling, interleukin-mediated inflammation, ECM organization and adhesion, growth factor/vascular signaling, and Wnt-related developmental signaling; 10 PPI modules were delineated. These findings indicate that nanomolar GenX elicits caspase-associated apoptotic response and oxidative stress in prostate stromal cells and provide a hypothesis-generating transcriptomic network map for targeted mechanistic follow-up of GenX-induced stromal toxicity.