PFOS exposure is linked to immune-metabolic disruption in MASLD: integrated population, computational, and experimental evidence.

Perfluorooctane sulfonate (PFOS), a persistent environmental pollutant, has been implicated in metabolic dysfunction-associated steatotic liver disease (MASLD), yet the underlying molecular mechanisms remain incompletely characterized. We constructed an integrated analytical framework combining population epidemiology, network toxicology, machine learning, transcriptomic analysis, single-cell mapping, molecular docking, and experimental validation. NHANES data (n = 1,834) were analyzed to assess the association between serum PFOS concentrations and FLI-defined MASLD. Machine-learning analyses using LASSO and SVM-RFE prioritized candidate genes from 874 overlapping PFOS-MASLD-associated genes. Single-cell RNA sequencing resolved cell-type-specific expression patterns, while molecular docking evaluated potential PFOS-protein interactions. A 12-week murine exposure model provided in vivo validation. Epidemiological analysis identified a nonlinear association between serum PFOS and MASLD odds (p < 0.001), with effects evident at background exposure levels (7.76 ng/mL). Convergent machine-learning analyses prioritized five candidate genes: CYP7A1, GRIA3, PHLDA1, SOCS2, and WNT5A. An exploratory five-gene model yielded an apparent AUC of 0.998 (95% CI 0.993-0.998) within the analyzed transcriptomic dataset. Single-cell analysis revealed cell-type specificity, with CYP7A1/PHLDA1 enriched in hepatocytes, SOCS2/WNT5A in hepatic stellate cells, and GRIA3 in T cells. Molecular docking predicted potential PFOS-protein interactions with the five candidate targets, with docking scores ranging from -6.3 to -9.3 kcal/mol. RT-qPCR analysis of PFOS-treated mouse liver showed transcriptional changes consistent with the computational predictions. PFOS-treated mice exhibited hepatic lipid accumulation, elevated liver injury markers, and lipid dysregulation. Together, these findings support an association between PFOS exposure and MASLD-related hepatic dysfunction and propose a hypothesis-generating immune-metabolic framework requiring prospective and direct mechanistic validation.