TCEP induces liver injury through suppression of the PI3K/AKT axis: Integrated evidence from epidemiology, network toxicology, and In vivo validation.

Tris(2-chloroethyl) phosphate (TCEP) is a widely used chlorinated organophosphate flame retardant, but its role in liver injury remains unclear. In this study, we integrated survey-weighted NHANES analysis, multi-source bioinformatics, and multi-dose in vivo validation to evaluate the association between TCEP exposure and liver injury. Higher TCEP exposure was associated with increased ALT, AST, GGT, and hepatic steatosis index (HSI) in NHANES 2011-2018 participants. These associations were more pronounced in overweight and obese individuals, suggesting increased metabolic susceptibility. Integrative analyses based on transcriptomic data, target prediction, pathway enrichment, machine learning, and immune infiltration profiling identified nine core targets potentially involved in TCEP-related liver injury, including PPARGC1A, STAT3, SRC, AKT1, CTNNB1, CDKN1A, PIK3R1, TP53, and SMAD3, and indicated suppression of PI3K/AKT signaling as a key mechanistic event. In a multi-dose mouse exposure model, TCEP-induced liver injury was confirmed by serum biochemical alterations and histopathological damage. Furthermore, immunofluorescence showed reduced P-AKT and PGC-1α signals together with enhanced P-SMAD3 and p53 expression, while Western blotting further verified inhibition of the PI3K/AKT pathway, activation of SMAD3/p53 signaling, and downregulation of PGC-1α. A calycosin intervention experiment further showed partial attenuation of TCEP-induced biochemical and histopathological liver injury, accompanied by restoration of PI3K/AKT phosphorylation. Overall, this study provides integrated epidemiological, bioinformatic, and experimental evidence that TCEP exposure is associated with liver injury and supports a candidate mechanistic framework involving PI3K/AKT-PGC-1α suppression and activation of SMAD3/p53-related injury-remodeling signaling in TCEP hepatotoxicity.