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Reducing Uncertainties in Quantitative AOPs by Analysis of Thyroid Hormone in the Neonatal Rat Brain
Citation:
Ford, J., C. Riutta, K. OShaughnessy, P. Kosian, I. Hassan, AND Mary E. Gilbert. Reducing Uncertainties in Quantitative AOPs by Analysis of Thyroid Hormone in the Neonatal Rat Brain. Society of Toxicology 2021 Virtual Annual meeting, Virtual, NC, March 12 - 26, 2021. https://doi.org/10.23645/epacomptox.14473662
Impact/Purpose:
Poster presented to the Society of Toxicology annual meeting March 2021
Description:
Adequate supplies of thyroid hormones (TH) are essential for brain development. Serum TH is routinely used in regulatory toxicology to identify chemicals with the potential to induce neurological deficits. Thus, serum TH occupies a pivotal position in adverse outcome pathways (AOP) for developmental neurotoxicity. Recent evidence, however, suggests that downstream neurological deficits do not always accompany reductions in serum TH. As such, TH levels in the fetal and neonatal brain may be necessary for the development of quantitative AOPs of thyroid-dependent neurotoxicity, yet technological challenges have limited their report. We optimized protocols for brain sample preparation to reduce matrix interference, enhance recoveries, and assess TH levels in neonatal rat brain to address this need. Male and female offspring from Long-Evans rats (n=5) were euthanized on postnatal days (PN) 0, 2, 6, and 14, and whole-brain or neocortex was collected. Isotopically-labeled internal and surrogate standards were added to 100 mg of brain homogenate, and TH were separated from lipids via a modified Folch extraction. Anion exchange solid-phase extraction further removed residual phospholipids. Quantification of TH was achieved by liquid chromatography-mass spectrometry using scheduled multiple reaction monitoring (sMRM) with a method detection limit of 0.005 ng/g for each TH. The mean recovery for all TH ranged from 70% to 125%. Linearity (r≥0.998) was observed from 0.005 ng/g to 25.6 ng/g for each TH. Based on surrogate standards, intra- and inter-day precisions were determined to be ± 15%. Brain TH concentrations increased over developmental time, T4 and T3 ranging from 0.560 to 3.05 ng/g and 0.900 to 5.00 ng/g, respectively, from PN0 to PN14. Only minor TH concentration differences were observed in perfused versus nonperfused samples, and no sex-dependent differences were evident in brain or serum TH concentrations at these ages. Incorporating brain TH measures in studies of developmental exposure to thyroid disrupting chemicals is feasible; improving potential neurotoxicity predictions will advance the utility of AOPs for risk determination. This work does not reflect US EPA policy.
URLs/Downloads:
DOI: Reducing Uncertainties in Quantitative AOPs by Analysis of Thyroid Hormone in the Neonatal Rat Brain
JERMAINE FORD SOT POSTER FY21 FINAL.PDF (PDF, NA pp, 514.828 KB, about PDF)