Developmental Hypothyroidism: An Underappreciated Risk Factor of Brain Barrier Disruption

Exposure to environmental thyroid disrupting chemicals is a concern for pregnant women and children as thyroid hormone (TH) action controls brain development. Recently, through RNA-sequencing (RNA-seq) and confocal microscopy, we showed that the blood-brain and blood-cerebrospinal fluid barriers may be affected in newborn rats with moderate hypothyroidism. This is consistent with what is observed in adult hypothyroid patients, where brain barrier function is compromised but rescued by TH supplementation. Thus, brain barrier function may be an overlooked target of endocrine disrupting chemicals. To address if TH action controls the development and/or function of the brain barriers, we gavaged pregnant rats with 0 or 0.875 mg/kg/day propylthiouracil (PTU) from gestational day (GD) 6 to postnatal day (PN) 14. This exposure significantly reduced dam and pup serum T4 and T3 during the first 2 postnatal weeks. Pup brain T4 and T3 were also significantly reduced. On PN8 we isolated pup choroid plexus (blood-cerebrospinal fluid barrier) and performed RNA-Seq. We detected 1102 differently expressed genes (DEGs) in the choroid plexus of PTU exposed animals as compared to controls (q<0.05). Five different claudins (tight junction proteins) were altered in the choroid plexus, including downregulation of claudin-5, which maintains brain barrier occluding activity. Gene Ontology analyses of the DEGs showed enrichment for the following biological processes: oxidative phosphorylation, ATP metabolic process, and cellular respiration. Ingenuity Pathway Analysis showed similar enrichment patterns, and also indicated increased oxidative stress and apoptosis. To investigate the functionality of the brain barriers, in vivo permeability assays were conducted using NHS-EZ-link biotin (443 Da). These experiments show that following cardiovascular perfusion, extraversion of the biotin marker into the brain parenchyma was observed in PTU exposed animals, while controls maintained this tracer within the brain’s vasculature. In summary, these data show that developmental hypothyroidism affects the transcriptome of the blood-cerebrospinal fluid barrier and the function of the blood-brain barrier. Thus, not only could environmental thyroid disrupting chemicals target the brain barriers, but a fetus, infant, and/or child with thyroid dysfunction may be more susceptible to other neurotoxicants due to increased barrier permeability. This work does not reflect US EPA policy.

Impact/Purpose

Exposure to environmental thyroid disrupting chemicals during pregnancy has been linked to several neurodevelopmental disorders. We recently showed that after inducing mild maternal hypothyroidism with propylthiouracil (PTU), newborn rat pups appeared to have brain barrier disruption. Brain barriers are specialized vascular structures that protect brain tissue and cerebrospinal fluid (CSF) from any harmful substances found in the blood. To further investigate the relationship between TH and the brain barriers, we performed RNA-sequencing (RNA-seq) of the choroid plexus (the blood-CSF barrier) and carried out functional experiments to test the permeability of the blood-brain barrier. Pregnant rats were dosed with either 0 or 0.875 mg/kg/day of PTU from gestational day (GD) 6 to postnatal day (PN) 14. We found 1102 differently expressed genes (DEGs) in the choroid plexus of PTU exposed offspring relative to controls on postnatal day 8. Using the bioinformatic programs Gene Ontology and Ingenuity Pathway Analysis, we found that these DEGs are responsible for oxidative phosphorylation, cellular respiration, and apoptosis, suggesting that hypothyroidism induces oxidative stress at the choroid plexus. Next, we performed cardiovascular perfusions using a tracer of a known molecular weight called NHS-EZ-link biotin (443 Da) in PN8 pups. Normally this 443 Da traces would be contained within the brain’s vasculature as the blood-brain barrier prohibits substances of this size from freely entering the brain tissue. However, we showed that PTU exposed demonstrated extraversion of this tracer into the brain tissue. Control animals contained this marker within the brain’s blood vessels as expected. Together, these data show that the blood-brain barrier is altered in PTU exposed animals. In summary, these data show brain barrier function can be affected by thyroid disrupting chemicals and hypothyroidism. With increased permeability of the brain barriers and compromised function altogether, fetuses and children have a higher risk of being exposed to other neurotoxicants. These data will help further the Agency’s interest in protecting childrens health and aid risk accessors in understanding the neurodevelopmental effects of thyroid disrupting chemicals. 

Citation

Sasser, A., K. Bell, J. Ford, M. Gilbert, AND K. O'Shaughnessy. Developmental Hypothyroidism: An Underappreciated Risk Factor of Brain Barrier Disruption. Society of Toxicology, Nashville, TN, March 19 - 23, 2023.