6:2 Cl-PFESA exposure and non-obstructive azoospermia: Insights from network toxicology and experimental validation

As a prominent replacement for conventional perfluorooctane sulfonate (PFOS), 6:2 chlorinated polyfluoroalkyl ether sulfonate (6:2 Cl-PFESA) has been widely used, raising health concerns. However, the precise toxicological impacts of this compound on the male reproductive system remain largely uncharacterized. To address this gap, we employed a comprehensive strategy integrating computational prediction, multi-omics analysis, and cell experiments to investigate potential molecular links between 6:2 Cl-PFESA exposure and non-obstructive azoospermia (NOA)-associated spermatogenic dysfunction. Among 977 putative target genes of 6:2 Cl-PFESA, 69 overlapped with NOA-associated differentially expressed genes (DEGs). Machine-learning analyses further identified DOT1L and AHCY as the most prominent candidate targets. Both genes were downregulated across independent NOA cohorts and were predominantly expressed in spermatocytes, spermatogonia, and spermatids according to single-cell transcriptomic analysis. Molecular docking suggested potential interactions between 6:2 Cl-PFESA and DOT1L or AHCY. Their expression was also associated with ssGSEA-derived enrichment scores for macrophages, regulatory T cells, and Th1 cells. In GC-1 spg cells, 6:2 Cl-PFESA exposure increased reactive oxygen species production and apoptosis and reduced DOT1L and AHCY protein levels. Overall, these findings suggest that DOT1L and AHCY may be involved in the response of spermatogonial cells to 6:2 Cl-PFESA exposure, thus providing a basis for further mechanistic investigation.