P-706 Integration of Network Toxicology and Molecular Docking Reveals Acetyl Tributyl Citrate-Induced Premature Ovarian Insufficiency: Mechanistic Insights into FoxO Signaling and Epigenetic Dysregulation

Abstract Study question Does acetyl tributyl citrate (ATBC) promote premature ovarian insufficiency through epigenetic dysregulation (EZH2/DNMT1/DNMT3A) and FoxO signaling pathway activation? Summary answer Acetyl tributyl citrate (ATBC) accelerates premature ovarian insufficiency through epigenetic dysregulation (EZH2/DNMT1/DNMT3A) and FoxO signaling pathway activation, leading to genomic instability and follicular impairment. What is known already Acetyl tributyl citrate (ATBC), a widely used plasticizer, impairs ovarian homeostasis by inducing oxidative stress, endocrine disruption, and epigenetic dysregulation, particularly through its interactions with DNMT1 and EZH2, which disrupt folliculogenesis and genomic stability. However, the mechanisms underlying ATBC’s synergistic effects with other endocrine disruptors and its long-term impact on ovarian reserve remain poorly characterized, limiting the development of therapeutic interventions for chemical-induced ovarian aging and premature ovarian insufficiency (POI). Study design, size, duration A multidisciplinary study combined network toxicology (ChEMBL, SwissTargetPrediction), molecular docking (AutoDock Vina), and in vivo validation in twenty 8-week-old female Sprague-Dawley rats (220 ± 20 g) were randomized into control and POI groups (n = 10 per group). POI was induced via 15-day oral ATBC exposure (200 mg/kg/day). Multi-omics analyses (GEO dataset, scRNA-seqs, qPCR) and ovarian PCR were conducted. The study spanned September 1–November 1, 2024, including 2-week acclimatization, 15-day exposure, and 3-week post-exposure analyses. Participants/materials, setting, methods This study utilized 8-week-old female Sprague-Dawley (SD) rats (220 ± 20 g, n = 10/group) and data from ChEMBL, SwissTargetPrediction, and GEO databases. In silico analyses (AutoDock Vina, STRING) and in vivo experiments (15-day ATBC exposure) were performed. Key methodologies included KEGG pathway enrichment, multi-omics analyses (GEO datasets, scRNA-seq), and qPCR of ovarian tissues to elucidate the molecular mechanisms underlying ATBC exposure. Main results and the role of chance Network toxicology identified 62 overlapping molecular targets between acetyl tributyl citrate (ATBC) and premature ovarian insufficiency (POI). GO/KEGG analyses revealed significant enrichment (p < 0.05) in cancer-related pathways and FoxO signaling, both of which are linked to ovarian dysfunction. Among 11 hub genes, three epigenetic regulators—EZH2, DNMT1, and DNMT3A—were significantly downregulated in POI, as validated by GEO datasets, single-cell RNA sequencing (scRNA-seq), and quantitative PCR (qPCR). DNMT3A suppression (p = 0.0225) impaired DNA methylation and genomic stability, while EZH2 downregulation (p = 0.0358; validation p = 0.00586) disrupted H3K27me3-mediated gene silencing, compromising follicular development. DNMT1 reduction (p = 0.00269) exacerbated ovarian aging. Molecular docking confirmed strong ATBC binding (ΔG < −7.0 kcal/mol) to these targets, suggesting direct interactions driving epigenetic dysregulation. Statistical significance across multi-platform analyses (p < 0.05) and false discovery rate (FDR) correction minimized false positives. However, biological variability in ovarian responses to ATBC and potential confounders (e.g., environmental factors) underscore the need for larger cohorts and clinical validation. Nevertheless, multi-modal validation approaches enhance the robustness of causal inferences. Limitations, reasons for caution This study is limited by small sample size (n = 10/group) and potential environmental confounders, which may affect reproducibility and generalizability. Biological variability in ovarian responses to ATBC and the absence of clinical validation necessitate larger, well-controlled studies to confirm findings and assess their translational relevance. Wider implications of the findings Exposure to ATBC (acetyl tributyl citrate) potentially accelerates ovarian aging through epigenetic dysregulation, underscoring its reproductive toxicity. These findings underscore the need for regulatory evaluation and further research on endocrine disruptors, emphasizing clinical validation and preventive measures, such as minimizing environmental exposure and developing targeted interventions to protect reproductive health. Trial registration number No