Assessing the hepatotoxicity of PFOA, PFOS, and 6:2 Cl-PFESA in black-spotted frogs (Rana nigromaculata) and elucidating potential association with gut microbiota

Pollution caused by per- and polyfluoroalkyl substances (PFASs) has become a major global concern. The association between PFAS-induced hepatotoxicity and gut microbiota in amphibians, particularly at environmentally relevant concentrations, remains elusive. Herein we exposed male black-spotted frogs (Rana nigromaculata) to 1 and 10 μg/L waterborne perfluorooctanoic acid (PFOA), perfluorooctanesulfonic acid (PFOS), and 6:2 chlorinated polyfluorinated ether sulfonate (6:2 Cl-PFESA) for 21 days; subsequently, liver histopathological, oxidative stress, molecular docking, gene/protein expression, and gut microbiome analyses were conducted. PFOS and 6:2 Cl-PFESA exposure enhanced serum alanine aminotransferase and aspartate aminotransferase activities, and markedly increased hepatic area of vacuoles and inflammatory cell infiltration, while PFOA exposure increased serum alanine aminotransferase but not aspartate aminotransferase activities and affected hepatic area of vacuoles and inflammatory cell infiltration to a lesser extent. All three PFASs elevated catalase, glutathione S-transferase, and glutathione peroxidase activities and glutathione and malondialdehyde contents in the liver, suggesting the induction of oxidative stress. Further, PFASs could bind to mitogen-activated protein kinases (p38, ERK, and JNK), upregulating not only their expression but also the expression of downstream oxidative stress-related genes and that of P-p38, P-ERK, and Nrf2 proteins. In addition, PFAS exposure significantly increased the relative abundance of Proteobacteria and Delftia and decreased that of Firmicutes and Dietzia, Mycoplasma, and Methylobacterium-Methylorubrum in the order of PFOS ≈ 6:2 Cl-PFESA > PFOA. Altogether, it appears that PFOS and 6:2 Cl-PFESA are more toxic than PFOA. Finally, microbiota function prediction, microbiota co-occurrence network, and correlation analysis between gut microbiota and liver indices suggested that PFAS-induced hepatotoxicity was associated with gut microbiota dysbiosis. Our data provide new insights into the role of gut microbiota in PFAS-induced hepatotoxicity in frogs.