Implications of an Improved Model of the TSH Receptor Transmembrane Domain (TSHR-TMD-TRIO).

The thyroid-stimulating hormone receptor (TSHR) is a G-protein-coupled receptor group A family member with 7 transmembrane helices. We generated 3 new models of its entire transmembrane region using a 600 ns molecular simulation. The simulation started from our previously published model, which we have now revised by also modeling the intracellular loops and the C-terminal tail, adding internal waters and embedding it into a lipid bilayer with a water layer and with ions added to complete the system. We have named this model TSHR-TMD-TRIO since 3 representative dominant structures were then extracted from the simulation trajectory and compared with the original model. These structures each showed small but significant changes in the relative positions of the helices. The 3 models were also used as targets to dock a set of small molecules that are known active compounds including a new TSHR antagonist (BT362), which confirmed the appropriateness of the model with some small molecules showing significant preference for one or other of the structures.

A simple short term method to study thyroid disruption using a fetal rat thyroid culture.

INTRODUCTION: Thyroid modulation activity has not been investigated for many chemical substances. Due to ethical, practical and financial reasons, in vivo evaluation of a large number of compounds is not feasible. It has been proposed that an in vitro mechanism-based strategy could be more adequate for the identification of thyroid hormone disrupting chemicals. Here we describe a simple and mostly inexpensive, short term culture assay to study thyroid disruption. METHODS: Fetal thyroids collected from gestation day 20.5 were cultured up to 24h in Hank's saline solution, at 37°C with oxygenation at 0 and 12h. Viability of the cultured explants was evaluated by the MTT assay. Positive (thyroid stimulating hormone, TSH) and negative (6-propyl-2-thiouracil, PTU) modulation of cultured thyroids was assessed with morphometrical analysis of H & E stained gland sections. Thyroxine expression was evaluated by immunohistochemistry. RESULTS: Viability was shown to increase with time of culture with higher metabolic activity being achieved at 24h as compared to shorter periods of incubation. Follicular epithelial cells exhibited a statistically significant dependence on thyrotropin concentration, although more evident in the inner than in the outer portion of the glands. As expected, TSH induced expression of thyroxin while PTU inhibited it. DISCUSSION: GD20.5 fetal thyroids may be cultured up to 24h under relatively simple laboratory conditions during which viability and function of the gland are preserved showing that it is possible to reproduce in vivo response under in vitro conditions. This culture could be a suitable short term assay to study mechanism of thyroid disruption.

Single-Photon Emission Computed Tomography for Preclinical Assessment of Thyroid Radioiodide Uptake Following Various Combinations of Preparative Measures.

BACKGROUND: MicroSPECT/CT imaging was used to quantitatively evaluate how iodide uptake in the mouse thyroid is influenced by (i) route of iodine administration; (ii) injection of recombinant human thyrotropin (rhTSH); and (iii) low iodide diet (LID) in euthyroid and triiodothyronine (T3)-treated mice. METHODS: Pertechnetate (99mTcO4-) and 123I thyroid uptake in euthyroid and T3-treated animals fed either a normal-iodine diet (NID) or an LID, treated or not with rhTSH, and radiotracer administered intravenously, subcutaneously, intraperitoneally or by gavage, were assessed using microSPECT/CT imaging. Western blotting was performed to measure sodium/iodide symporter expression levels in the thyroid. RESULTS: Systemic administration of radioiodide resulted in a higher (2.35-fold in NID mice) accumulation of iodide in the thyroid than oral administration. Mice fed LID with systemic radioiodide administration showed a further two-fold increase in thyroid iodide uptake to yield a ∼5-fold increase in uptake compared to the standard NID/oral route. Although rhTSH injections stimulated thyroid activity in both euthyroid and T3-treated mice fed the NID, uptake levels for T3-treated mice remained low compared with those for the euthyroid mice. Combining LID and rhTSH in T3-treated mice resulted in a 2.8-fold higher uptake compared with NID/T3/rhTSH mice and helped restore thyroid activity to levels equivalent to those of euthyroid animals. CONCLUSIONS: Systemic radioiodide administration results in higher thyroidal iodide levels than oral administration, particularly in LID-fed mice. These data highlight the importance of LID, both in euthyroid and T3-treated, rhTSH-injected mice. Extrapolated to human patients, and in the context of clinical guidelines for the preparation of differentiated thyroid cancer patients, our data indicate that LID can potentiate the efficacy of rhTSH treatment in T3-treated patients.

Therapeutic drug monitoring during pregnancy and lactation: thyroid function assessment in pregnancy-challenges and solutions.

The diagnosis and monitoring of thyroid disease necessitates the knowledge of thyroid pathophysiology and of the technical limitations of current thyroid-related biochemical tests. Thyroid disease diagnosis and monitoring are further complicated during pregnancy and lactation, due to pregnancy-related changes in thyroid hormone metabolism. Dramatic changes that occur in thyroxine and triiodothyronine ranges during pregnancy pose challenges for hypothyroid gravidas. Very early in pregnancy, levothyroxine replacement needs to be increased. Moreover, increases in thyroid hormone replacement need to be conducted individually and on a timely basis. For reasons that are still not entirely clear, although dependent in part on changes in thyroxine binding, free thyroxine (FT4) levels decrease as pregnancy progresses necessitating the use of trimester-specific reference intervals for appropriate replacement. Thyroxine binding protein levels vary by hormonal status, inheritance, and disease states and are higher in pregnancy; hence, FT4 assays became popular because they measure the unbound hormone. However, current FT4 immunoassays are estimate tests that do not reliably measure FT4 and are known to be sensitive to alterations in binding proteins and therefore are method-specific. The need to reliably identify hypothyroxinemic pregnant patients, especially in the first trimester, is of prime importance for early fetal brain development before the fetal thyroid functions. This article addresses 1) the current limitations of laboratory-free thyroxine immunoassay methodologies and especially during pregnancy; 2) trimester-specific reference intervals for thyroid function tests; and 3) the study of levothyroxine pharmacokinetics in pregnant and nonpregnant women.

[Comparison of administration of rhTSH with withdrawal of thyroid hormone. Follow-up of patients with differentiated thyroid carcinoma]. / Estudio comparativo entre la utilización de TSH recombinante y la estimulación endógena con TSH. Valoración en el seguimiento de los pacientes con carcinoma diferenciado de tiroides.

UNLABELLED: Recombinant human thyrotropin (rhTSH) has been introduced recently in follow up of differentiated thyroid cancer (DTC) patients, as an alternative of thyroid hormone withdrawal. The aim of this retrospective study is to compare recombinant human thyrotropin versus endogenous stimulation. MATERIAL AND METHODS: Thirty-three patients with DTC with previous thyroidectomy and thyroid ablation were selected. All patients underwent whole-body radioiodine scanning and third day serum thyroglobulin (TG) measurement by two techniques, the first one after conventional thyroid hormone withdrawal (TSHe, TGe), and the second one after rhTSH stimulation (TSHr, TGr). Measurement of TG was performed on the third day due to the infrastructure. We only included patients with stable disease, without therapeutic interventions between two consecutive controls in an interval inferior to one year. Two qualitative categories were defined for TG (positive TG > 2 ng/ml or negative TG<2 ng/ml) and whole-body radioiodine scan (positive or negative). RESULTS: TSHe: 62.9 +/- 55.48; TSHr: 113.16 +/- 50.6; (p: ns); TGe: 62.5 +/- 115.7; TGr: 54.6 +/- 111.1; (p: 0.044). Quantitative data analysis showed significant differences between two techniques. Qualitative data analysis showed no significant differences in clinical setting based in TG and radioiodine scan. CONCLUSIONS: Administration of rhTSH produces a significantly higher increase of TSH than thyroid hormone withdrawal and lower increase in TG levels. There were no significant differences in the stage of disease (TG and whole-body radioiodine scan).

Recombinant human thyroid-stimulating hormone: initial bioactivity assessment using human fetal thyroid cells.

We have assessed the bioactivity of newly available recombinant human TSH (rec-hTSH) using human fetal thyroid cells, with the longer term aim of assessing its use for clinical applications. Rec-hTSH caused a consistent and dose-related increase in thyroid monolayer cell cAMP release and human thyroglobulin (hTg) secretion, confirming its bioactivity. Repetitive studies (n = 5) allowed us to derive an estimated biopotency for the rec-hTSH preparation examined of 5.6 IU/mg compared to 10 IU/mg for commercially available bovine TSH for human use. The rec-hTSH had a bioimmune ratio of 0.55, similar to that of purified pituitary hTSH standards, Furthermore, rec-hTSH induced thyroid epithelial cell growth, as evidenced by a decrease in thyroid cell doubling time from 54 +/- 2.1 to 31 +/- 1.7 h (P less than 0.005). Hence, rec-hTSH is a potent glycoprotein hormone preparation when measured in a homologous human thyroid cell culture system. Rec-hTSH could serve as a future definitive International Standard and has the potential for a useful diagnostic and therapeutic reagent.

Alterations in thyroidal economy in a systemic illness induced by turpentine oil injection to the rat.

Injection of turpentine oil (5 microliter/g, sc) to Sprague Dawley rats was associated with a significant reduction in serum concentration of T4, T3 and TSH that lasted throughout 48 h of study. Dialyzable fraction of T4 and resin uptake of T3 did not change for 10 h after turpentine oil injection, but were increased significantly at 24 h and 48 h. Serum rT3 concentration was decreased significantly at 24 h and 48 h. The pituitary content of TSH in the experimental rat was significantly increased at 24 h after turpentine oil injection, but the TSH-beta subunit content was decreased significantly. Serum TSH response to TRH did not change in experimental rats. Thyroidal radioiodine uptake, serum T4, and serum T3 in experimental rats demonstrated little or no increase in response to exogenous bovine TSH. A thyroid hormone binding inhibitor (THBI) was detected in serum and iodothyronine 5'-monodeiodinating activity was decreased significantly in liver, kidney and heart of rats injected repeatedly with turpentine oil. Oxygen consumption of experimental rats did not change appreciably. Our data suggest that administration of turpentine oil to the rat leads, within a few hours of the injection, to a systemic illness associated with marked changes in thyroidal economy.

Assessment of the biopotency of anti-thyroid drugs using porcine thyroid cells.

The effect of six drugs on the uptake and organification of iodide by porcine thyroid cells stimulated with bovine TSH (10 miU/L) has been investigated. The drugs fall into two categories: the peroxidase inhibitors, methimazole (MMI), 2-thiouracil (2-TU) and 3-amino 1,2,4 triazole (3-ATA) and the ionic inhibitors, lithium chloride (LiCl), potassium perchlorate (KC10(4], and sodium iodide (NaI). All the drugs led to a dose-related inhibition of iodide metabolism. The most potent effect on iodide uptake was seen with NaI which inhibited this function by 20% even at 10(-8) mol/l. In contrast, the most potent effect on iodide organification was observed with methimazole which led to a 25% inhibition at 10(-8) mol/l. The concentrations of drug which gave rise to a 50% inhibition of iodide uptake were (mumol/l) 0.26 (NaI), 3.5 (KClO4), 9.7 (2-TU), 15 (MMI), 26 (3-ATA), and 1500 (LiCl). The comparable figures for organification were 0.13 (MMI), 0.18 (2-TU), 0.23 (NaI), 1.2 (3-ATA), 15 (KClO4) and 1300 (LiCl). We conclude that this in vitro system has considerable potential for the assessment of potency and possible bioassay of anti-thyroid drugs of varying structures and sites of action.

Assessment of thyrotoxicity using in vitro cell culture systems.

The potential thyrotoxic effect of SK&F 93479 (an H2-receptor antagonist), aminoglutethimide (AG) and glutethimide were studied by examining their effects on 125iodide uptake and organification (incorporation into thyroglobulin) in cultured thyroid cells from the pig and rat. These effects were compared with those of positive controls (3-isobutyl-1-methylxanthine (IBMX), methimazole (MTI), propylthiouracil (PTU) and NaClO4). SK&F 93479 had no effect on 125I uptake in rat FRTL-5 cells or on 125I uptake and organification in porcine thyroid cells. In contrast, MTI, PTU and NaClO4 all depressed 125I uptake and organification and IBMX potentiated 125I uptake. It is concluded, therefore, that SK&F 93479 does not increase 125I uptake in rats in vivo by a direct action on the thyroidal iodide trapping mechanism. AG, but not glutethimide, depressed 125I organification by cultured porcine thyrocytes without affecting uptake. These results suggest that AG is thyrotoxic in rats and man probably as a result of its ability to inhibit iodide organification in thyroid cells.

Assessment of thyroid functional reserve in the cat by the thyrotropin-stimulation test.

Serum thyroxine (T4) concentrations before and after various IV doses of bovine thyrotropin (TSH) were measured over a 48-hour period in 19 healthy cats. Base-line T4 values, as measured by radioimmunoassay, varied greatly. The peak T4 concentration occurred 6 hours after TSH injection, and there was an increase in post-TSH serum T4 concentration that was linearly related to the logarithm of the dose. Greatest stimulation was seen with the highest dose used (1 U of TSH/kg of body weight), and 6 hours after administration of this dose, the serum T4 concentration range was 4.1 to 8.4 micrograms/dl. The post-TSH serum T4 concentration and the absolute increase in serum T4 concentration after TSH administration correlated more closely with the TSH dose than did the ratio of post-TSH serum T4 concentration to base-line T4 concentration. Therefore, in cats with normal thyroid-binding protein concentrations, the former indices should represent the most reliable assessment of thyroid functional reserve.

A re-assessment of the effect of thyrotrophin (TSH) on the tibial plate bioassay for growth hormone (GH).

The claim has been made that thyrotrophin (TSH) can augment the action of growth hormone (GH) to stimulate growth of the epiphysial cartilage plate of the hypophysectomized rat's tibia. The TSH induces its effect via secretion of thyroid hormones which in turn enhance the stimulatory action of GH. If this is true then the employment of the tibia test, whose endpoint is the increase in thickness of the epiphysial cartilage plate in response to GH present either in crude pituitary extracts or relatively purified preparations, which also are likely to contain modest or appreciable quantities of TSH, requires further examination. The present study utilized various fractions of crude pituitary extracts from intact and thyroidectomized rats that respectively contained appreciable quantities of GH or essentially no GH. Fractional aliquots of pituitary extracts from thyroidectomized rats were administered concomitantly with graded doses of exogenous GH to hypophysectomized rats to determine the point at which TSH in the extracts was sufficiently able to stimulate significant tibial plate growth when compared to recipients given GH alone. Purified GH and TSH were also administered in various doses to hypophysectomized recipients in a further attempt to delineate the dose range at which TSH augments the action of GH to promote significant chondrogenesis of the epiphysial plate. The results indicate that the enhancement of the GH effect on the cartilage plate by TSH was evident only when quantities above 100 microng bovine GH were co-administered with 100 mU bovine TSH. As little as 40 mU TSH augmented the growth effect of 400 microng GH on the cartilage plate, demonstrating that smaller quantities of TSH could potentiate larger quantities of GH. These data, therefore, suggest that extracts equivalent to not more than one-half of a normal adult rat's anterior pituitary gland should be administered to hypophysectomized rats for bioassay of GH. Fractions of glands greater than this may contain sufficient amounts of TSH to augment the appreciable quantities of GH already present.

A radioimmunoassay for 3,3',5'-L-triiodothyronine (reverse T3): assessment of thyroid gland content and serum measurements in conditions of normal and altered thyroidal economy and following administration of thyrotropin releasing hormone (TRH) and thyrotropin (TSH).

The present report describes the development of a radioimmunoassay for 3,3',5'-L-triiodothyronine (reverse T3) which is performed on unextracted serum. Utilizing this radioimmunoassay, 21 normal subjects had a mean (+/-SD) serum reverse T3 level of 60 +/- 12 ng/100 ml, 17 of 19 hyperthyroid patients had elevated serum reverse T3 levels, and 10 of 11 hypothyroid subjects had decreased serum reverse T3 concentrations. Thyroidal secretion of reverse T3 was assessed by measurements in samples obtained from the internal carotid artery and jugular vein of sheep following the administration of thyrotropin releasing hormone (TRH) or bovine thyrotropin (TSH). Reverse T3 levels were increased 45-60 min after TRH administration, but TSH administration produced inconsistent alterations in reverse T3, although 18 of 27 samples obtained after TSH injection were higher than their average respective baseline concentration and the mean peak reverse T3 level was 14% higher than baseline. Following TRH administration to 10 normal human subjects, mean serum reverse T3 levels significantly increased from 53.6 ng/100 ml to 56.3ng/100 ml (P less than .05). The thyroid gland content of reverse T3 in human autopsy material was 6.5 +/- 1.5 microng/g tissue. Both pregnancy and estrogen administration were associated with increases in serum reverse T3 concentrations presumably because of their ability to augment thyroxine binding globulin synthesis.