Impact of PFAS exposure on the fecal metabolome

Abstract: The human gut microbiota plays a crucial role in human health and therefore imbalances in gut microbiota functioning can lead to the development of metabolic disorders. Short-chain fatty acids (SCFA), tricarboxylic acid (TCA) cycle metabolites, bile acids (BA) and other metabolites are the essential compounds for the metabolome. Per- and Polyfluoroalkyl substances (PFAS) are a group of chemicals that can be absorbed onto the lumen by food. Since many PFAS has shown to possess a long list of adverse effects on human health, the short term impact of PFAS on gut microbiota functioning using an in vitro model that mimics fermentation in the human colon was explored. Samples obtained from in vitro fecal fermentation were then analysed through high-performance liquid chromatography coupled to time-of-flight mass spectrometry (LC-qToF-MS) using targeted and non-targeted approaches. Fecal samples were donated by four donors which were put through a fermentation over 24 hours and treated with no PFAS mixture (control), low concentration mixture of PFAS (PFAS-L) and high concentration mixture of PFAS (PFAS-H). The PFAS mixture contained perfluorooctanesulfonic acid (PFOS), perfluorooctanoic acid (PFOA), perfluorohexanesulfonic acid (PFHxS), perfluorononanoic acid (PFNA) and perfluorodecanoic acid (PFDA). For the fermentation, samples were collected after 0, 2, 4, 6, 8 and 24 hours. The samples were spun down and the supernatant was collected. Two approaches were applied to explore the metabolism in the fermentation extracts: The first one was a “targeted SCFA and TCA cycle metabolites analysis’’ and the second one was “untargeted analysis of polar and nonpolar metabolites’’. Results show that all TCA cycle metabolites displayed different trends with the compound and little to no variation between the treatments. However, although there was no significant difference, 4 core compounds of the TCA metabolism were lower after treatment with PFAS-L or PFAS-H. For the untargeted method, 78 of 2855 compounds were identified. When comparing control samples, PFAS-H and PFAS-L, 200 features showed statistically significant differences. Most of these had higher concentrations for PFAS-treated samples. When PFAS-H was compared only with PFAS-L, 30 features were found to be statistically significant, indicating that distinct concentrations of PFAS differentially affect gut microbiota metabolism. For future identification, further analysis must be done, preferably with MS/MS, to obtain more structural information for these significant metabolites, since the masses of the fraction ions are needed to narrow down the search in databases used to obtain the identity of an ion. PFAS and the concentration seem to have an impact on the gut microbiota, the study should be done with additional donors to archive trends applicable to a whole population.