Citation:

Riutta, C., I. Hassan, J. Ford, P. Kosian, Katherine OShaughnessy, S. Degitz, AND M. Gilbert. Thyroid Hormone Measurements in Rat Brain by Mass Spectrometry. Developmental Neurotoxicology Society (DNTS), San Diego, CA, June 23 - 26, 2019.

Impact/Purpose:

Quantitative adverse outcome pathways (qAOPs) are being developed for the neurodevelopmental consequences of thyroid disruption. Serum hormones are currently used as a flag for potential developmental neurotoxicity, but the magnitude and developmental timing of that change have not been adequately defined. The lack of clear and sensitive markers of developmental neurotoxicity linked directly to thyroid hormone disruption has impeded our ability to build predictive qAOP models. In the current AOP, the key event of serum hormone reductions is directly linked to decrements in brain hormones. Brain hormones have been lacking from the literature but represent a more metric more closely aligned with potential neurotoxicity. Defining the quantitative relationships between serum and brain hormones will provide a scientific foundation on which to interpret serum hormone data routinely measured in regulatory studies. Due to technical limitations, measuring TH in developing brain tissue is not common, largely due to a lack of methods to isolate THs from a complex tissue matrix, and instrumentation capable of quantifying low concentrations of THs. The work described in this abstract details the ontology of brain hormones in brains of neonatal rat pups using a highly sensitive mass spectrometry analytical method developed in NHEERL MED laboratory and implemented here in RTP in the Analytical Core. We are very encouraged as the data show that the method is sufficiently sensitive to measure brain TH in pups on the day of birth and that brain TH, like serum TH, increases with age. The within study variability is within acceptable ranges but work needs to be done to identify factors that contribute variability across studies.

Description:

Thyroid hormones (THs) are essential for the growth and development of the brain. Due to technical limitations, measuring TH in developing brain tissue is not common; largely due to a lack of methods to isolate THs from a complex tissue matrix, and instrumentation capable of quantifying low concentrations of THs. Recently, a sensitive method to assess THs in rat brain (detection limits of 50 pg/mL for T3 and T4) using liquid-liquid extraction with liquid chromatography/mass spectrometry (LC/MS/MS) was developed at US EPA. We present here a preliminary evaluation of TH measures in control rats using this methodology. Our goals were four-fold: 1) determine if brain TH of neonatal rats could be measured with this analytical method; 2) identify age-controlled ranges in brain concentrations of TH; 3) examine within study biological variability across ages; 4) document study-to-study variability. Brain samples from 3-4 studies were analyzed within each age group (n=3-5 samples/age/study). All samples were well above the detection limits of the instrument and a clear ontological progression in brain TH concentrations was evident. Averaged across studies, brain T4 increased from 0.422, 1.20, 2.11, to 2.86 ng/g from postnatal days (PN) 0, 2, 6 and 14, respectively. Average T3 concentrations were generally higher than that obtained for T4, and also increased with age from 0.740, 1.31, 2.48, to 5.74 ng/g from PN0 to PN14. Coefficients of variation (CV) of brain T4 and T3 within individual studies were commonly below 10% at any given age. The CV between studies, however was substantially higher for both analytes, exceeding 50% in some cases. We anticipate that inclusion of additional studies, continued improvements in tissue harvest/extraction procedures, and assessment using the same analytical instrument will reduce the variations between studies. In the 3 most recent studies where these factors were controlled, lower CVs of 20% and 5.9% for T4 and T3 were obtained for the same historical control (HC) sample run over 7 analytical runs. Further work to identify and control factors to limit variability is needed, but low within study CVs and stable HC indicate reliable estimates of brain TH are attainable.

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