Effects of Gestational and Lactational Exposure to Perfluorohexanoic Acid on the cerebellar transcriptome

Legacy 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 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 ddH20 using treat based administration. Given the high PFHxA levels in cerebellum and protracted developmental window, acute transcriptional dysregulation and cellular morphology of the cerebellum was assessed on the last day of exposure at P21. Using bulk-RNA sequencing, overall PFHxA showed subtle effects in 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. Overall design: C57Bl6J pregnant mice were exposed to control, 0.32 mg/kg of bw (lower), or 50 mg/kg of bw (higher) exposures of PFHxA from gestational day (GD) 0 through postnatal day (P) 21 resulting in in utero and lactational exposure to PFHxA. On P21, offspring were euthanized and whole cerebella were dissected and prepared for bulk-RNA sequencing.