Copper Pyrithione Inhibits Trophoblast Cell Function: Mechanisms Underlying Its Embryotoxic Potential

Copper pyrithione (CuPT), a widely used antimicrobial agent in antifouling coatings and consumer products, has potential reproductive toxicity. This study aims to evaluate the impact of CuPT on human trophoblast-like JEG-3 cells and elucidate potential molecular mechanisms. Proliferation was assessed by CCK-8; migration and invasion by wound-healing and Matrigel Transwell assays; cell cycle and apoptosis by flow cytometry; intracellular reactive oxygen species (ROS) by DCFH-DA; DNA double-strand breaks by γ-H2AX immunofluorescence. Transcriptomic changes were profiled by RNA-seq with differential expression and GO enrichment analyses. Autophagy and endoplasmic reticulum (ER) stress markers were assessed by immunofluorescent staining. Key differentially expressed genes were validated by qRT-PCR. CuPT suppressed JEG-3 proliferation in a time- and concentration-dependent manner and reduced migratory and invasive capacities versus controls. Under 80 nM for 72 h, cell-cycle phase distribution showed no significant changes, but late apoptosis increased and intracellular ROS levels rose, indicating oxidative stress. γ-H2AX foci were elevated, consistent with DNA damage. RNA-seq revealed 648 upregulated and 451 downregulated genes, with enrichment of ER-stress, unfolded-protein response, intrinsic apoptotic signaling, and autophagy pathways among upregulated genes, and suppression of oxygen-response, glycolytic/energy metabolism, sterol biosynthesis, and cell-adhesion/mitotic regulation among downregulated genes. Concurrently, CuPT activated pronounced autophagosome formation and an ER stress response. CuPT treatment downregulated CYTB, ND6, and Egln3 expressions and upregulated NUPR1 and SQSTM1 expressions. CuPT impairs trophoblast-like JEG-3 cell proliferation, migration, and invasion, accompanied by increased ROS and DNA damage. Integrated RNA-seq and staining data indicate activation of ER stress and autophagy.