Mutations of thyrotropin receptor gene

 Thyrotropin is the primary pituitary hor- mone which stimulates the growth and differentiation of thyroid cells. TSH binds a specific receptor present in the plasma membrane of thyroid cells and signals the G protein transducers, which activate different effec- tors, mainly adenyl cyclase and phospholipase C. The TSH receptor belongs to a broad class of receptors known as seven-loop receptors because they contain a long stretch of amino acids which cross the plasma membrane seven times. Mutations in the TSH receptor gene have been found in hyperfunctioning thyroid adenomas. These mutations are: (a) somatic (present only in the tumor), (b) dominant (only one copy of the gene is affected), and (c) lead to the constitutive activation of the cAMP signaling cascade. Most mutations which have been identified occur in the intracellular loop III and in the transmembrane domain VI. Germline mutations in the same regions of the receptor have been found in congenital nonautoimmune hyperthyroidism. In addition, germ line mutations have been described in the extracellular domain of the receptor leading to increased TSH levels. The clinical implications of these findings are discussed. Received: 15 January 1996 / Accepted: 8 March 1996     

Thyroglobulin inhibition of thyrotropin binding to thyroid plasma membrane.

The effect of thyroglobulin (TG) on binding of TSH to thyroid plasma membranes was studied in vitro. Human and bovine thyroid plasma membranes have specific binding sites for bovine [125I]TSH. The binding of [125I]TSH is inhibited by the addition of purified TG (100 ng/microgram/ml). The inhibitory mechanism appears to be noncompetitive when subjected to Lineweaver-Burk analysis. Incubation of TG with TSH did not show an interaction, as assessed by sucrose gradient centrifugation. Plasma membranes prepared from human thyroid tissue have specific binding sites for human TG, as shown by [125I]TG binding assay. The TG binding was not affected by adding low concentrations of unlabeled bovine TSH. In the presence of very high concentrations of TSH, TG binding was increased. Hemoglobin, beta-lactoglobin, and ovalbumin did not have an inhibitory effect on [125I]TSH and [125I] TG binding to membrane preparations. Both [125I]TSH and [125I]TG binding were inhibited by 10 mM neuraminic acid. These results suggested that 1) TG released from thyroid gland may have a regulatory effect on TSH binding to its specific receptor, and 2) there are specific binding sites for TG on the thyroid plasma membrane.     

Pre-operational survey of a U mine near Val Vedello, Italy

Results obtained from radiometric research carried out from 1980-81 in the region of U mines in the Val Vedello are provided. Data are presented on 222Rn, 226Ra and natural U concentrations in spring and surface waters. Concentrations were found to be relatively low. There is some increase in the vicinity of the exploratory U mine but this is, at least up to the present, only of a local character. Data are presented on external gamma exposure, 210Pb and 210Po in foodstuffs and in the urine of normal and exposed population as well as 226Ra in milk and vegetable samples. The aim of this study is to outline a preliminary map of natural radioactivity on the site before starting the mining activity. This will enable us later to detect changes caused by the U mining, milling and mill-tailing disposal operations.     

Growing thyroid nodules with benign histology and RET rearrangement

Some benign thyroid nodules are stationary in size over time while others grow progressively, indicating that there is a broad individual variability within benign nodules. To date, it is very difficult to predict if a benign thyroid nodule will grow in size and which will be its trend over time. While BRAF(V600E) is a highly specific marker of thyroid cancer, RET rearrangements have been disclosed also in non malignant thyroid lesions and their biological significance is debated. We compared the clinical history of three histologically benign thyroid nodules harboring RET rearrangements with that of 6 benign nodules bearing wild type RET. The nodules negative for RET rearrangements were followed for 10 years by ultrasonographic evaluation, showing a slow, constant enlargement. Three patients with benign nodules diagnosed at FNAC, were followed for 11, 9 and 7 years by annual ultrasonographic evaluation. After several years of latency, the nodules had an unexpected and gradual increase in their dimensions, reaching a large final size. A second FNAC confirmed the previous cytologic diagnosis of benign lesion. Because of the increasing size of the nodules, the patients were advised to surgery. Before undergoing thyroidectomy, we performed molecular diagnostic tests that revealed the absence of BRAF(V600E) and the presence of RET/PTC-1 in one nodule and RET/PTC-3 in the two others. Despite the presence of this oncogene, the samples were histologically classified as benign hyperplastic nodules. These findings lead us to speculate that histologically benign hyperplastic thyroid nodules containing RET rearrangements might represent a subgroup of nodules with a rapid size increase.     

Amiodarone induces cytochrome c release and apoptosis through an iodine-independent mechanism

Amiodarone (AMD) is one of the most effective antiarrhythmic drugs available. However, its use is often limited by side-effects, mainly hypo- or hyperthyroidism. As AMD displays direct toxic effect on different cell types, we investigated the cytotoxic effect of AMD and its main metabolite, desethylamiodarone (DEA), in thyroid (TAD-2) and nonthyroid (HeLa) cell lines. Both AMD and DEA displayed a dose-dependent toxicity in TAD-2 and HeLa cells, although DEA was more effective. Both TAD-2 and HeLa cells underwent apoptosis, as evidenced by plasma membrane phosphatidylserine exposure and DNA fragmentation. Inhibition of protein synthesis with cycloheximide and inhibition of endogenous peroxidase activity with propylthiouracil did not affect this AMD- and DEA-induced apoptosis in TAD-2 cells. Western blot analysis did not display variations in the expression of p53, Bcl-2, Bcl-XL, and Bax proteins during the treatment with AMD and DEA. Generation of reactive oxygen species, investigated by flow cytometry with dichlorofluorescein diacetate, did not show the production of free radicals during drug treatment. Furthermore, Western blot analysis of cytosolic and mitochondrial fractions prepared from AMD-treated cells demonstrated that AMD induces the release of cytochrome c into the cytosol from the mitochondria. These data indicate that AMD induces cytochrome c release from mitochondria, triggering apoptosis through an iodine-independent mechanism, and that this process is not mediated by modulation of p53, Bcl-2, Bcl-XL, or Bax protein expression and does not involve the generation of free radicals.     

Cardiovascular safety of acute recombinant human thyrotropin administration to patients monitored for differentiated thyroid cancer

Eleven patients who had undergone total thyroidectomy for differentiated thyroid cancer and who were on chronic TSH-suppressive therapy with levothyroxine (L-T4), underwent 24-h Holter electrocardiogram and Doppler-echocardiography before and after acute recombinant human TSH (rhTSH) administration for disease staging. The treatment, which was generally well tolerated, did not affect circulating thyroid hormones levels, nor did it have measurable effects on heart rate, rhythm, left ventricular morphology, or systo-diastolic function. Notably, arterial blood pressure tended to be slightly reduced after rhTSH administration, although in no instance did the patients become frankly symptomatic. Our data demonstrate that rhTSH does not alter cardiovascular function acutely. Consequently, it can safely be used in the routine staging of patients affected by differentiated thyroid cancer.     

Percutaneous ethanol injection under Power Doppler ultrasound assistance in the treatment of autonomously functioning thyroid nodules

Power Doppler (PD) is a recent color-Doppler Ultrasound (US)-technique, which allows to detect the presence of flow even in very small vessels, providing a sort of angiographic micromap. The aim of this study was to evaluate whether percutaneous ethanol injection (PEI) outcome might be improved by injecting the ethanol into the nodule under PD assistance. Thus, 14 patients affected with pretoxic (PTA) and 8 with toxic adenoma (TA) were submitted to this alternative tool. Before PEI, all patients were submitted to a careful endocrinological study, including an US-guided fine-needle biopsy in order to exclude the presence of malignancy. In addition, all the nodules were evaluated at PD-US and their vascular patterns were recorded on videotape and compared with those obtained after treatment. The procedure consisted of slow injection of sterile ethanol under direct PD-US control. The number of PEI sessions was 2.3+/-0.1 in PTA and 3.0+/-0.3 in TA. All patients were also evaluated 3, 6, 12 and 18 months after PEI. Successful therapy was considered when normalization of thyroid hormones and TSH was achieved together with the disappearance of nodular hyperactivity and complete recovery of extra-nodular tracer uptake at scintigraphy. PEI was tolerated very well by all patients. The most common side effect was a transient local or irradiated pain. All patients with PTA and 6 out of 8 patients with TA were successfully treated. In these cases, PD-US showed the progressive reduction of the intranodular blood flow, up to its extinction after 6-12 months, with the presence of little perilesional vascular spots. Nodular shrinkage was obtained in all patients (from 4.7+/-0.7 to 1.1+/-0.4 ml in PTA and from 21.0+/-2.8 to 6.2+/-1.6 ml in TA). In conclusion, PD assistance improves PEI procedure, since it allows to guide the ethanol injection towards the principal afferent vessels of the nodules and to monitor the diffusion and the effects of ethanol on nodular vascularization.     

Iodide excess induces apoptosis in thyroid cells through a p53-independent mechanism involving oxidative stress

Thyroid toxicity of iodide excess has been demonstrated in animals fed with an iodide-rich diet; in vitro iodide is cytotoxic, inhibits cell growth, and induces morphological changes in thyroid cells of some species. In this study, we investigated the effect of iodide excess in an immortalized thyroid cell line (TAD-2) in primary cultures of human thyroid cells and in cells of nonthyroid origin. Iodide displayed a dose-dependent cytotoxicity in both TAD-2 and primary thyroid cells, although at different concentrations, whereas it had no effect on cells of nonthyroid origin. Thyroid cells treated with iodide excess underwent apoptosis, as evidenced by morphological changes, plasma membrane phosphatidylserine exposure, and DNA fragmentation. Apoptosis was unaffected by protein synthesis inhibition, whereas inhibition of peroxidase enzymatic activity by propylthiouracil completely blocked iodide cytotoxicity. During KI treatment, reactive oxygen species were produced, and lipid peroxide levels increased markedly. Inhibition of endogenous p53 activity did not affect the sensitivity of TAD-2 cells to iodide, and Western blot analysis demonstrated that p53, Bcl-2, Bcl-XL, and Bax protein expression did not change when cells were treated with iodide. These data indicate that excess molecular iodide, generated by oxidation of ionic iodine by endogenous peroxidases, induces apoptosis in thyroid cells through a mechanism involving generation of free radicals. This type of apoptosis is p53 independent, does not require protein synthesis, and is not induced by modulation of Bcl-2, Bcl-XL, or Bax protein expression.