Citation:

Gilbert, M., J. Ford, C. Riutta, K. OShaughnessy, AND P. Kosian. Reducing Uncertainties in Quantitative Adverse Outcome Pathways by Analysis of Thyroid Hormone in the Neonatal Rat Brain. TOXICOLOGICAL SCIENCES. Society of Toxicology, RESTON, VA, 193(2):192-203, (2023). https://doi.org/10.1093/toxsci/kfad040

Impact/Purpose:

Background/Overview:  Thyroid hormones are essential for brain development. A wide variety of manmade chemicals have the potential to disrupt the thyroid axis. Most xenobiotics are identified as potential thyroid disrupting chemicals by their action to reduce levels of thyroid hormone in the blood of laboratory animals, however the exact relationship between serum hormone changes and brain function remains unknown. Regulatory bodies have embraced the Adverse Outcome Pathway (AOP) Framework to assist in translating serum hormone changes to predict adverse neurodevelopmental outcomes stemming from exposure to thyroid disrupting chemicals. Brain thyroid hormones derive from direct transport of hormones from the blood to the brain. Once in the brain, thyroid hormones induce transcription of families of genes that control neurodevelopmental processes. As such, reductions in brain hormone concentrations represent a proximal intermediary in the pathway from serum hormone to thyroid hormone action that drives these thyroid-modulated neurodevelopmental processes. Assessment of this key event - brain hormone - will aid in the construction of AOPs and evaluation of potential neurotoxicity of thyroid disrupting chemicals. We have developed an assay to measure thyroid hormones in brain tissue from fetal and neonatal rats.  Relevancy to EPA Program/Regional Research Needs/Priorities: This research addresses Chemical Safety for Sustainability (CSS) Adverse Outcome Pathway (AOP Research Area 4.2). Name(s) of Program Office Reviewer(s) of Earlier Drafts:  N/A Program Office/Regional Office Co-Authors: N/A Description: Measuring thyroid hormones in the brain is challenging due to the rich phospholipid matrix that comprising brain tissue. The goal of the present study was to create a method for brain thyroid hormone measurement. We report a solid-phase extraction method using a mixed-mode strong anion exchange cartridge that retains the acidic functional group of thyroid hormones, permitting more aggressive washes, reducing phospholipid breakthroughs, and lowering detection limits. This optimized method was implemented to assess brain thyroid hormone concentrations over a range of ages in neonatal rats in tissue mass of ~50mg commensurate with the mass of a fetal rat brain. Major Observations and Results:   LCMS procedures were optimized to measure thyroid hormone in rat brain. Hormone recovery was enhanced by reducing phospholipid breakthrough using established liquid-liquid extraction procedures, an anion exchange column, a stringent column wash, and improved evaporation procedures. In combination, these procedures produced reliable age-dependent measures of brain T4 and T3. Minimal detection and quantification limits were determined. Quality control measures revealed excellent recovery and consistency.  Impact/Potential Implications of the Findings:  This study provides a methodology for reliably assessing brain thyroid hormones in neonatal rat brain. Brain hormones assessment is the most proximal downstream key event in a quantitative AOP for thyroid disruption and neurodevelopmental insult. Measures of brain thyroid hormone represent a needed linkage between the much relied upon measures of serum hormone to detect thyroid disrupting chemicals. Brain hormone measures provide a means to determine the quantitative relationships between hormones in serum and brain and to track how they vary over developmental time. Findings Advancing Existing Scientific Knowledge:  The findings provide a quantitative method to reliably assess concentrations of thyroid hormone in neonatal rat brain. This provides a means to assess if  these measures are perturbed by exposure to environmental contaminants with thyroid action. They provide a much needed bridge between serum and brain hormone disruption.  Publication Information. Scientific peer reviewed journal Contact:  Jermaine Ford, MSc, ORD, CTTE ford

Description:

A number of xenobiotics interfere with thyroid hormone (TH) signaling. Although adequate supplies of TH are necessary for normal brain development, regulatory reliance on serum TH as proxies for brain TH insufficiency is fraught with significant uncertainties. A more direct causal linkage to neurodevelopmental toxicity induced by TH-system disrupting chemicals is to measure TH in the target organ of most concern, the brain. However, the phospholipid-rich matrix of brain tissue presents challenges for TH extraction and measurement. We report optimized analytical procedures to extract TH in brain tissue of rats with recoveries >80% and low detection limits for T3, rT3, and T4 (0.013, 0.033, and 0.028 ng/g, respectively). Recovery of TH is augmented by enhancing phospholipid separation from TH using an anion exchange column coupled with a stringent column wash. Quality control measures incorporating a matrix-matched calibration procedure revealed excellent recovery and consistency across a large number of samples. Application of optimized procedures revealed age-dependent increases in neonatal brain T4, T3, and rT3 on the day of birth (postnatal day, PN0), PN2, PN6, and PN14. No sex-dependent differences in brain TH were observed at these ages, and similar TH levels were evident in perfused versus non-perfused brains. Implementation of a robust and reliable method to quantify TH in the fetal and neonatal rat brain will aid in the characterization of the thyroid-dependent chemical interference on neurodevelopment. A brain- in addition to a serum-based metric will reduce uncertainties in assessment of hazard and risk on the developing brain posed by thyroid system-disrupting chemicals.

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