Perfluorohexanesulfonic acid (PFHxS) induces oxidative stress and causes developmental toxicities in zebrafish embryos

Perfluorohexanesulfonic acid (PFHxS) is a short-chain perfluoroalkyl substance widely used to replace the banned perfluorooctanesulfonic acid (PFOS) in different industrial and household products. It has currently been identified in the environment and human bodies; nonetheless, the possible toxicities are not well-known. Zebrafish have been used as a toxicant screening model due to their fast and transparent developmental processes. In this study, zebrafish embryos were exposed to PFHxS for five days, and various experiments were performed to monitor the developmental and cellular processes. Liquid chromatography-mass spectrometry (LC/MS) analysis confirmed that PFHxS was absorbed and accumulated in the zebrafish embryos. We reported that 2.5 µM or higher PFHxS exposure induced phenotypic abnormalities, marked by developmental delay in the mid-hind brain boundary and yolk sac edema. Additionally, larvae exposed to PFHxS displayed facial malformation due to the reduction of neural crest cell expression. RNA sequencing analysis further identified 4643 differentiated expressed transcripts in 5 µM PFHxS-exposed 5-days post fertilization (5-dpf) larvae. Bioinformatics analysis revealed that glucose metabolism, lipid metabolism, as well as oxidative stress were enriched in the PFHxS-exposed larvae. To validate these findings, a series of biological experiments were conducted. PFHxS exposure led to a nearly 4-fold increase in reactive oxygen species, possibly due to hyperglycemia and impaired glutathione balance. The Oil Red O’ staining and qPCR analysis strengthens the notions that lipid metabolism was disrupted, leading to lipid accumulation, lipid peroxidation, and malondialdehyde formation. All these alterations ultimately affected cell cycle events, resulting in S and G2/M cell cycle arrest. In conclusion, our study demonstrated that PFHxS could accumulate and induce various developmental toxicities in aquatic life, and such data might assist the government to accelerate the regulatory policy on PFHxS usage.