High-Throughput Single-Cell Mass Spectrometry Reveals Sex-Specific Metabolic Responses to 6:2 Chlorinated Polyfluoroalkyl Ether Sulfonate in Zebrafish Liver Cells

Compound 6:2 chlorinated polyfluoroalkyl ether sulfonate (6:2 Cl-PFESA) is an emerging per- and polyfluoroalkyl substance (PFAS) with potential toxicity and health risks to biosystems and ecosystems. Here, we developed a metabolomics method based on single-cell mass spectrometry to investigate the hepatotoxicity and heterogeneous responses in zebrafish exposed to 6:2 Cl-PFESA. Zebrafish were exposed to an environmentally relevant concentration (200 ng/L) of 6:2 Cl-PFESA for 14 days. The livers were dissociated and prepared as cell suspensions and then introduced to high-throughput single-cell mass spectrometry for analysis of 6:2 Cl-PFESA and endogenous metabolites in individual primary liver cells. Significant sex-specific heterogeneity in 6:2 Cl-PFESA accumulation was observed (<i>p</i> < 0.05). Metabolomics analysis revealed perturbations in lipid metabolism, particularly affecting unsaturated fatty acids, ether lipids, and sphingolipids in zebrafish liver cells, indicating potential hepatotoxicity. Sex-dependent metabolic responses were evident: males showed notable changes in glucose and fatty acid metabolism, whereas females experienced pronounced disruptions in glycerophospholipid and amino acid pathways. ROC analysis identified sex-specific biomarkers, including FA(18:3) and FA(16:1) in males (AUC > 0.85), as well as proline and phosphatidylcholine in females (AUC > 0.90). These findings reflect metabolic dysregulation and highlight sex-specific responses. This study demonstrates the feasibility of single-cell metabolomics to elucidate the cellular mechanisms and metabolic responses of pollutant exposure, offering insights into precise and comprehensive toxicity assessments at the single-cell level.