Reducing uncertainties in quantitative adverse outcome pathways by analysis of thyroid hormone in the neonatal rat brain

Abstract

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.

Keywords: adverse outcome pathway; brain; mass spectrometry; thyroid hormones.

Figure 1.

Representative chromatogram of unlabeled thyroid hormones T3, rT3, and T4, injected on column at a (A) low amount (0.125 pg) and a (B) high amount (32 pg). Peaks for each hormone analyte are clearly resolved between 2.0 and 3.0 minutes. The chromatogram on the left with low intensity range on the y-axis shows that the signal-to-noise ratio is greater than 3.

Figure 2.

Investigation of phospholipids reduction in brain matrix from three different extraction procedures. Phosphatidylcholine was used as a readout of phospholipids using precursor ion scanning 184 m/z product ion. Solid-phase extraction with the Evolute Express AX cartridge (green) with the addition of a 2% formic acid in dichloromethane wash outperformed the other SPE cartridges, virtually eliminating phospholipid breakthrough.

Figure 3.

Calibration curves were generated by spiking solvent and matrix-matched samples with labelled T3, rT3 and T4 at concentrations ranging from 0.005 ng/g- 25ng/g. Measured calibration points were within ± 20% of their predicted value. The coefficient of determination, r2 for all curves was ≥ 0.995. The surrogate solvent curve closely approximated the matrix matched curve.

Figure 4.

A) To assess the suitability of the methods optimized using the Biotage AX-60 exchange column to reliably detect hormones in the brains of young rat pups, forebrains collected from rat pups (n=6) on postnatal day 2 were assessed for thyroid hormones. T3 and T4 were detected in the 1–2 ng/g range and rT3 at much lower concentration, but well above the minimal detectable limit of 0.033ng/g. Quality control is exemplified in the summary of recoveries in analytical runs across a range of studies conducted with this method as shown in B) for ¹³C₁₂-T3 and C) for ¹³C₁₂-T4. In each analysis only 2 of 44 runs (4.5%) fell outside the lower control limits of 80%.

Figure 5.

Serum and brain thyroid hormones in neonatal rat pups. A) Serum collected from male and female pups was pooled at each age. T3 and T4 increased with age (n=5/age group). B) Brain T3 and T4 increased with age and no differences were seen between perfused vs non-perfused brains (n=5–7/group). C) No significant differences in brain T3 or T4 were seen between male and female pups at the ages tested.