Reducing Uncertainties in Quantitative Adverse Outcome Pathways by Analysis of Thyroid Hormone in the Neonatal Rat Brain
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.