Perfluorohexyloctane (F6H8) is a semifluorinated alkane used in ophthalmic surgery as a vitreoretinal tamponade. It has long been considered biologically inert because of its chemical stability and presumed resistance to metabolism. However, emerging evidence from studies of structurally related fluorinated and non-fluorinated hydrocarbons suggests that such compounds can accumulate in the liver and undergo metabolic transformation. As the liver is a central organ for xenobiotic metabolism and lipid homeostasis, disruptions in hepatic metabolic pathways may have systemic consequences. Despite increasing clinical usage of F6H8, data on its potential metabolic effects and biotransformation in the human-relevant system remain poorly understood. Clarifying whether F6H8 induces metabolic reprogramming or forms biologically active metabolites is therefore critical for informed safety evaluation, particularly in the context of growing regulatory scrutiny of fluorinated compounds.
This study, published in Environmental Research, aimed to comprehensively characterize the metabolic effects of F6H8 exposure in human hepatocytes using untargeted metabolomics and lipidomics. Specifically, the study investigated whether F6H8 induces concentration-dependent metabolic alterations at the non-cytotoxic exposure levels, identified potential biotransformation products of F6H8, and assessed how both parent compound and any detected metabolites are linked with disruptions in amino acid, lipid, and central metabolic pathways in hepatocytes and their extracellular environment.
Human HepaRG hepatocytes were exposed for 24 hours to a range of non-cytotoxic concentrations of F6H8 selected to approximate estimated hepatic burdens resulting from repeated ophthalmic use. Comprehensive metabolomic and lipidomic profiling was performed on intracellular extracts and extracellular culture media using high-resolution liquid chromatography-mass spectrometry (LC-MS). Statistical analyses involved correlation analyses in exposure concentrations, differential abundance analysis relative to controls, and partial correlation network analysis to evaluate changes in the lipid class associations. Unknown compounds strongly correlated with F6H8 exposure were investigated by using MS/MS fragmentation, retention time prediction, and ion mobility mass spectrometry to support the structural annotation. Pathway enrichment and functional analyses were conducted to detect metabolic pathways perturbed at the lowest and highest exposure concentrations, which capture potential non-monotonic dosage response effects.
F6H8 exposure induced pronounced, concentration-dependent, and frequently non-monotonic metabolic changes in hepatocytes and extracellular media. A strong linear correlation between exposure concentrations and many detected compounds was seen, which includes 3 features absent in control samples. One of these was tentatively detected as perfluorohexyloctanoic acid, a carboxylic acid derivative of F6H8 based on accurate mass characteristics, MS/MS fragmentation patterns, retention time alignment, logP-based retention time prediction, and ion mobility-derived collision cross-section measurements. This result gives evidence that F6H8 may undergo oxidative biotransformation in the human hepatocytes. More than 130 metabolites and lipids showed major correlations with F6H8 concentration. Lipidomic analysis showed extensive changes in lipid profiles with elevated lipid levels in the extracellular medium, which suggests improved lipid efflux or membrane remodeling. Polyunsaturated fatty acids were generally upregulated with increasing exposure.
Saturated fatty acids and acylcarnitines were downregulated at the lowest exposure concentration and partially reversed or upregulated at higher concentrations. Pathway analyses showed that lipid metabolism, sphingolipid signaling, and glycosaminoglycan degradation pathways were specifically sensitive at low exposure levels. Higher concentrations disrupted amino acid metabolism, nitrogen handling, and central carbon metabolism. The biotransformation product perfluorohexyloctanoic acid showed significantly broader and stronger metabolic links than parent compound correlations over 280 intracellular metabolites and lipids. Pathways uniquely linked with this metabolite involved purine metabolism, bile acid metabolism, and mitochondrial flux-related pathways, which suggest improved bioactivity relative to F6H8.
In conclusion, this study evaluates that F6H8 exposure induces substantial metabolic alterations in human hepatocytes, including disruptions in lipid metabolism and amino acid processing. The tentative identification of a PFAS-like carboxylic acid biotransformation product challenges the assumption that semifluorinated alkanes are biologically stable. While the findings stem from in vitro studies and do not directly establish human health effects, they increase concerns about the systemic safety of F6H8 and emphasize the need for further in vivo studies to assess potential chronic effects associated with repeated exposure.
Reference: Alijagic A, Rotander A, et al. Metabolic effects and biotransformation of perfluorohexyloctane (F6H8) in human hepatocytes: comprehensive metabolomic and lipidomic profiling. Environ Res. 2026;S016041202600070X. doi:10.1016/j.envres.2026. 110112
