Acetyl tributyl citrate as a new risk factor for osteoporosis: An integrated study from multi-level toxicological assessment and experimental validation.

This study aimed to elucidate the toxicological effects and underlying mechanisms of the plasticizer acetyl tributyl citrate (ATBC) on osteoporosis (OP) development by integrating computational biology with experimental validation. Potential targets of ATBC and OP-related genes were retrieved from public databases, identifying 113 common targets. Robust machine learning modeling pinpointed five core genes (EGFR, SYK, BRD4, PTK2B, ADAMTS5), which demonstrated strong binding affinity with ATBC in molecular docking and exhibited stable binding in 100-ns molecular dynamics simulations. Furthermore, these genes correlated significantly with altered immune cell infiltration. Experimentally, ATBC exposure inhibited the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs), downregulating key markers (Runx2, OPN, OCN, OSX) and reducing alkaline phosphatase activity and mineralization. In vivo, ATBC induced significant bone loss in rats, characterized by decreased bone volume fraction (BV/TV), trabecular thickness (Tb.Th), and increased trabecular separation (Tb.Sp). In conclusion, this study demonstrates that chronic exposure to the plasticizer ATBC disrupts bone metabolic homeostasis by directly interacting with key targets, inhibiting osteogenic differentiation of BMSCs, and inducing deterioration of bone microstructure, ultimately promoting osteoporosis development. These findings reveal, for the first time, the substantial osteotoxic risk of ATBC, challenging its conventional safety profile and highlighting the imperative for stricter regulation of its environmental exposure and further investigation into its long-term health impacts.