Towards a science-based testing strategy to identify maternal thyroid hormone imbalance and neurodevelopmental effects in the progeny—part III: how is substance-mediated thyroid hormone imbalance in pregnant/lactating rats or their progeny related to neurodevelopmental effects?

This review investigated which patterns of thyroid- and brain-related effects are seen in rats upon gestational/lactational exposure to 14 substances causing thyroid hormone imbalance by four different modes-of-action (inhibition of thyroid peroxidase, sodium-iodide symporter and deiodinase activities, enhancement of thyroid hormone clearance) or to dietary iodine deficiency. Brain-related parameters included motor activity, cognitive function, acoustic startle response, hearing function, periventricular heterotopia, electrophysiology and brain gene expression. Specific modes-of-action were not related to specific patterns of brain-related effects. Based upon the rat data reviewed, maternal serum thyroid hormone levels do not show a causal relationship with statistically significant neurodevelopmental effects. Offspring serum thyroxine together with offspring serum triiodothyronine and thyroid stimulating hormone appear relevant to predict the likelihood for neurodevelopmental effects. Based upon the collated database, thresholds of ≥60%/≥50% offspring serum thyroxine reduction and ≥20% and statistically significant offspring serum triiodothyronine reduction indicate an increased likelihood for statistically significant neurodevelopmental effects; accuracies: 83% and 67% when excluding electrophysiology (and gene expression). Measurements of brain thyroid hormone levels are likely relevant, too. The extent of substance-mediated thyroid hormone imbalance appears more important than substance mode-of-action to predict neurodevelopmental impairment in rats. Pertinent research needs were identified, e.g. to determine whether the phenomenological offspring thyroid hormone thresholds are relevant for regulatory toxicity testing. The insight from this review shall be used to suggest a tiered testing strategy to determine whether gestational/lactational substance exposure may elicit thyroid hormone imbalance and potentially also neurodevelopmental effects.</p

Stereological assessment of the mean number of TH⁺ neurons in the SNpc following PQ or MPTP treatment in C57BL/6J and C57BL/6NHsd male mice.

<p>Five different groups (G1-G5) of animals, varying in age and site of experiment were injected with saline, paraquat or MPTP and the extent of TH⁺ neuron loss was assessed by design-based or model-based stereology. ** significantly different from control mice (p ≤ 0.01). Syngenta-sourced PQ was used to treat mice G1 to G3 mice, whereas Sigma Chemical PQ was used to treat mice in groups G4 and G5.</p

Pathological assessment of SNpc and striatum in PQ- or MPTP-treated mice.

<p>Microscopic appearance of the SNpc (G-L) and striatum (A-F) in control (column 1), PQ- (column 2) or MPTP-treated (column 3) mice, 48 hours after dosing. Tyrosine hydroxylase (TH) immunostaining was decreased in the striatum (C) and SNpc (I) of the MPTP-treated animal but was unchanged in the PQ-treated mouse (B, H) compared to the control (A, G). Amino cupric silver (AmCuAg) staining was used to reveal degenerating neurons in the SNpc (J-L) and degenerating fibers in the striatum (D-F). There were no differences in AmCuAg staining in either the SNpc or striatum after PQ treatment compared to the control (D,J). AmCuAg staining was increased in the SNpc and striatum of the MPTP-treated mouse (F,L). The scale bar shown in Panel L represents 400 μm in panels A-C, G-I and 40 μm in panels D-F and J-L.</p

Appearance of microglia and astrocytes in SNpc and striatum of PQ- and MPTP-treated mice.

<p>Microscopic appearance of Iba-1-stained microglia (A-F) and GFAP-stained astroctyes (G-L) in the SNpc of mice 48 hours after PQ (column 2) or MPTP treatment (column 3) compared to a control (column 1). Iba-1 immunostaining of microglia was increased in the MPTP-treated mouse (C, F) but was not different in the PQ-treated mouse (B, E) compared to the control (A, D). Similarly, increased GFAP immunostaining of astrocytes was noted in the MPTP-treated mouse (I, L) but not in the PQ-treated mouse (H, K) compared to the control (G, J). Scale bar A-L 40 μm.</p

Mean histopathological severity scores in control, paraquat and MPTP-treated groups of C57BL/6J male mice.

<p>Mice were 16 weeks of age at the time of treatment initiation. Mice were administered 10 mg/kg/dose PQ·Cl₂ by ip injection, twice a week for 3 weeks and were sacrificed 8, 16, 24, 48, 96 or 168 hours after the last dose. Control mice were given the vehicle while MPTP-treated mice received four injections of MPTP (16 mg/kg/dose; expressed as free base) at 2-hour intervals, and then euthanized 48 hours after the final dose. Serial sections through the SNpc were evaluated qualitatively and the group mean severity grades are plotted. Grades 0 to 5 reflect increasing intensity of staining for Iba-1, AmCuGg, GFAP and decreased staining intensity of TH.</p

Correlation between the number of activated microglia in the SNpc and the number of TH⁺ neurons.

<p>The correlation between the number of activated microglia in the SNpc and the number of TH⁺ neurons was assessed in the same animal in two studies on C57BL/6J mice (A, B) and one study using C57BL/6NHsd (C) performed at two different sites (WIL, A) and SJCRH (B, C). A significant negative inverse correlation between TH⁺, DA neuron number and activated microglia was observed in MPTP-treated mice, while no correlation was seen in PQ- or saline treated mice.</p

Number, design and location of studies examining effects of paraquat and MPTP on C57BL/6J and C57BL/6NHsd male mice (aged 9 or 16 weeks)¹.

<p>Number, design and location of studies examining effects of paraquat and MPTP on C57BL/6J and C57BL/6NHsd male mice (aged 9 or 16 weeks)<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164094#t001fn001" target="_blank">¹</a>.</p