Gestational and Lactation Exposure to Perfluorohexanoic Acid Results in Sex-Specific Changes in the Cerebellum in Mice

Currently regulated per- and polyfluoroalkyl substances (PFAS) have been associated with immune, endocrine, and neurotoxicity following gestational exposures. As a result, industries have effectively replaced them with next-generation PFAS, including perfluorohexanoic acid (PFHxA). PFHxA is increasingly found in the serum of pregnant women and in breast milk, and adult human post-mortem studies indicate that PFHxA is found in the brain, with the highest concentrations in the cerebellum and hypothalamus. Despite evidence of gestational, lactational, and nervous system exposure to PFHxA, developmental neurotoxicity (DNT) testing in mammals has not been conducted. For DNT evaluation, we exposed pregnant C57Bl/6J mice daily from gestational day 0 through postnatal day (P) 21 to two PFHxA exposure levels (a lower (0.32 mg/kg of body weight (bw), or higher (50 mg/kg of bw) dose of PFHxA)) or ddH₂O using treat-based administration. Given the high PFHxA levels in the cerebellum in post-mortem studies and the cerebellum's protracted developmental window, we assessed acute transcriptional dysregulation and cellular morphology in this brain region on the last day of exposure at P21. Using bulk-RNA sequencing, we found that PFHxA exposure had subtle effects on transcripts related to neurons and glia, with females having a greater number of dysregulated transcripts than males. Using immunohistochemistry, we found that Purkinje cell linear frequency was increased in specific lobules in the higher-exposure group and that microglial morphology underwent subtle changes in specific cerebellar layers in the lower-exposure group in both sexes. Together these data suggest that PFHxA exposure may have lobule-specific impacts on the development of both neurons and glia in the cerebellum, highlighting the importance of studying the neurotoxicity of PFHxA in both sexes.