A Novel Screening Framework Integrating a Two-Cell Assay System and Computational Toxicology to Identify Disruptors of the GnRH Neuron-Gonadotrope Axis

The hypothalamic-pituitary-gonadal (HPG) axis is a major target of endocrine-disrupting chemicals (EDCs) and a central regulator of sexual maturation and reproduction in humans and vertebrates. The gonadotropin-releasing hormone (GnRH) neuron-gonadotrope axis (GG axis) is a core component within the HPG system, linking upstream and downstream hormonal cascades. In this study, we developed a two-cell assay system using independently cultured GT1-7 and LβT2 cell lines to simulate GG axis hormonal regulation and evaluate the disruptive effects of 50 environmental chemicals on the expression of key hormones and receptors genes, including GnRH, G protein-coupled receptor 54 (GPR54), GnRH receptor (GnRHR), chorionic gonadotropin α (CGα), luteinizing hormone β (LHβ), and follicle-stimulating hormone β (FSHβ). Using the Toxicological Priority Index (ToxPi) tool, we quantified and weighted alterations in these six target genes, ranking the chemicals according to their GG axis disruption potential and identifying high-risk EDCs. We further validated this integrated high-throughput screening system by assessing the <i>in vivo</i> effects of hexafluoropropylene oxide trimer acid (HFPO-TA), the third-ranked compound and a widely used perfluorooctanoic acid (PFOA) substitute, on pubertal onset timing in female mice, confirming the system's effectiveness and accuracy. This comprehensive strategy provides a reliable framework for efficient identification of GG axis disruptors and offers a novel, streamlined approach to chemical risk assessment.