6:2 Chlorinated Polyfluoroalkyl Ether Sulfonate (6:2 Cl-PFAES), a PFOS Alternative, Impairs Testosterone Synthesis in Leydig Cells by Targeting Lipophagy via the SIRT1–FOXO1–RAB7 Axis: An In Vitro and In Vivo Study

As an emerging alternative to perfluorooctanesulfonate (PFOS), 6:2 chlorinated polyfluoroalkyl ether sulfonate (6:2 Cl-PFAES) is associated with male reproductive disorders, yet its testicular targets and mechanisms remain unclear. This study combined in vivo exposure of male C57BL/6J mice (0, 4, 40, and 400 μg/L for 18 weeks) and in vitro assays in primary Leydig cells to demonstrate that 6:2 Cl-PFAES dose-dependently reduced sperm count and serum/testicular testosterone, accompanied by testicular interstitial vacuolation. Transcriptomics and metabolomics demonstrated the concurrent significant enrichment of autophagy pathways and disruption of lipid metabolism. Mechanistically, 6:2 Cl-PFAES suppressed expressions of Sirtuin 1 (SIRT1), ras-related GTP binding protein 7 (RAB7), and deacetylation of forkhead box class O1 (FOXO1), thereby impairing lipophagy flux and promoting lipid droplets accumulation. Molecular docking indicated comparable binding affinity of 6:2 Cl-PFAES and PFOS to SIRT1 (−10.4 kcal/mol), and further research demonstrated that 6:2 Cl-PFAES reduced the deacetylation activity of SIRT1. Crucially, Sirt1 overexpression in transgenic mice (Sirt1 TG ) or pharmacological activation of SIRT1 (SRT1720) and RAB7 (ML-098) rescued testosterone synthesis and restored lipophagy flux. This study identifies the SIRT1–FOXO1–RAB7 axis as a core target for 6:2 Cl-PFAES-induced suppression of testosterone biosynthesis via lipophagy dysregulation, providing critical molecular insights for assessing the reproductive risks of PFOS alternatives.