Per- and polyfluoroalkyl substances (PFAS), manmade chemicals that accumulate in the body over time, have been linked to liver disease and cancer, but it is not yet clear how they cause damage. USC researchers used a lab model of the human liver to analyze changes at the cellular level, finding that some PFAS triggered fat accumulation and others caused cell damage linked to cancer. The researchers used spheroids, sophisticated 3D models that recreate the structure of the liver using cells from 10 human donors (five male and five female). They exposed the spheroids to four types of PFAS commonly found at high levels in the blood: perfluorooctanoic acid (PFOA), perfluorohexanesulfonic acid (PFHxS), perfluorooctanesulfonic acid (PFOS) and perfluorononanoic acid (PFNA). Each chemical was tested separately to determine its specific effects on liver cells. After seven days of exposure, the researchers separated the spheroids into individual cells for analysis. They used single-cell RNA sequencing to analyze gene expression and a dye-based method to measure fat buildup in the spheroids under a microscope. All four PFAS interrupted cell signaling and immune functions, but exact changes varied from one chemical to the next. Both PFOA and PFHxs increased fat accumulation—PFOA by causing cells to produce more fat and PFHxS by causing cells to retain fat. Both PFOS and PFNA triggered cancer-related changes in cells, but PFNA had a stronger effect, increasing activity in cellular pathways related to inflammation, oxidative stress and DNA repair. Of the cells exposed to PFNA, 61.3% showed gene changes linked to cancer. The researchers also found that liver cells from male and female donors responded differently to PFAS exposure. PFOA has stronger effects on female liver cells, while PFOS had stronger effects on cells from male donors.