Screening for drugs potentially interfering with MCT8-mediated T3 transport in vitro identifies dexamethasone and some commonly used drugs as inhibitors of MCT8 activity.

BACKGROUND: Monocarboxylate transporter 8 (MCT8) is the first thyroid hormone transporter that has been linked to a human disease. Besides genetic alterations other factors might impair MCT8 activity. AIM: This study aimed at investigating whether some common drugs having a structural similarity with TH and/or whose treatment is associated with thyroid function test abnormalities, or which behave as antagonists of TH action can inhibit MCT8-mediated T3 transport. METHODS: [125I]T3 uptake and efflux were measured in COS-7 cells transiently transfected with hMCT8 before and after exposure to increasing concentrations of hydrocortisone, dexamethasone, prednisone, prednisolone, amiodarone, desethylamiodarone, dronedarone, buspirone, carbamazepine, valproic acid, and L-carnitine. The mode of inhibition was also determined. RESULTS: Dexamethasone significantly inhibited T3 uptake at 10 µM; hydrocortisone reduced T3 uptake only at high concentrations, i.e. at 500 and 1000 µM; prednisone and prednisolone were devoid of inhibitory potential. Amiodarone caused a reduction of T3 uptake by MCT8 only at the highest concentrations used (44% at 50 µM and 68% at 100 µM), and this effect was weaker than that produced by desethylamiodarone and dronedarone; buspirone resulted a potent inhibitor, reducing T3 uptake at 0.1-10 µM. L-Carnitine inhibited T3 uptake only at 500 mM and 1 M. Kinetic experiments revealed a noncompetitive mode of inhibition for all compounds. All drugs inhibiting T3 uptake did not affect T3 release. CONCLUSION: This study shows a novel effect of some common drugs, which is inhibition of T3 transport mediated by MCT8. Specifically, dexamethasone, buspirone, desethylamiodarone, and dronedarone behave as potent inhibitors of MCT8.

Electric and magnetic fields do not modify the biochemical properties of FRTL-5 cells.

BACKGROUND: Electric and magnetic fields (EMF) might be involved in human disease and numerous research and scientific reviews have been conducted to address this question. In particular thyroid structural and functional alterations caused by various forms of non-ionizing radiation have been described. AIM: The aim of this study was to analyze the possible effects of EMF on thyroid, in particular we analyzed the effects caused by a GSM (Global System for Mobile Communications) signal (900 MHz) on cultured thyroid cells (FRTL- 5). MATERIAL AND METHODS: The experimental setup was designed in order to expose samples to a radiofrequency wave in well-controlled conditions. We used the FRTL-5 cell line, an epithelial monoclonal continuous cell line derived from Fisher rat thyroid tissue growing as monolayer, expressing the TSH receptor and the sodium-iodide symporter (NIS). FRTL-5 were subsequently irradiate for 24, 48, and 96 h with EMF (800-900 MHz, power-frequency of mobile communication systems) and iodide uptake and cAMP production were measured. RESULTS: The irradiation of cells with EMF at 900 Mhz for 24, 48, and 96 h did not influence the level of cAMP production and was not able to modify iodide accumulation in FRTL- 5 cells with respect to basal conditions. CONCLUSIONS: In conclusion, EMF do not seem to be able to interfere with the biochemical properties of FRTL-5 cells in vitro.

Study of potential inhibitors of thyroid iodide uptake by using CHO cells stably expressing the human sodium/iodide symporter (hNIS) protein.

BACKGROUND: Thyroid gland is highly dependent on dietary intake of iodine for normal function, so it is particularly subjected to "endocrine disruptor" action. The human sodium/iodide symporter (hNIS) is an integral plasma membrane glycoprotein mediating the active transport of iodide into thyroid follicular cells, a crucial step for thyroid hormone biosynthesis. Beyond to perchlorate and thyocianate ions a few other inhibitors of iodide uptake have been described. AIM: The aim of this study was to investigate if 10 substances usually used as drugs in clinical practice were able to inhibit NIS-mediated iodide uptake in vitro. MATERIALS AND METHODS: A CHO cell line stably expressing hNIS was used to test any inhibition of NIS-mediated iodide uptake exerted by drugs. Perchlorate and thyocianate ions were used as positive controls. RESULTS: None of the analyzed substances was able to significantly inhibit iodide uptake in our system. As we expected, perchlorate and thyocianate ions were able to inhibit iodide uptake in a dose-dependent manner. CONCLUSIONS: In conclusion, we carried out an in vitro assay to evaluate the potential inhibitory effect of common drugs on NISmediated iodide uptake by using CHO-hNIS cells. None of the analyzed substances was able to inhibit iodide uptake; only perchlorate and thyocianate were able to inhibit iodide uptake in a dose-dependent manner.

Patients affected by vitiligo and autoimmune diseases do not show antibodies interfering with the activity of the melanocortin 1 receptor.

BACKGROUND: Vitiligo is an acquired depigmenting disorder characterized by the loss of melanocytes from the epidermis with the development of white patches in various distribution. The pathogenesis of vitiligo is still unknown, but the association with autoimmune disorders and organ specific autoantibodies, supports the hypothesis of an autoimmune pathogenesis. AIM: The aim of the present study was to investigate if autoantibodies present in sera of patients affected by vitiligo may be able to interfere with the activity of the αMSH on the melanocortin 1 receptor (MC1R). MATERIALS/ SUBJECTS AND METHODS: IgG from the sera of 41 patients with vitiligo associated or not with thyroid autoimmune diseases or other autoimmune pathologies were incubated with HBL20 cells (human malignant melanocytes expressing the MC1R) in the presence of a sub-maximal dose of αMSH. A normal IgG range was determined by using IgG extracted from 30 control sera of normal subjects. RESULTS: None of the IgG from vitiligo patients was able to inhibit αMSH-stimulated cAMP production in HBL20 cells. CONCLUSIONS: Autoantibodies against MC1R are rare or absent in sera of vitiligo patients.

Effects of a thyroid-stimulating human monoclonal autoantibody (M22) on functional activity of LH and FSH receptors.

OBJECTIVE: The glycoprotein hormones luteinizing hormone (LH), follicle-stimulating hormone (FSH), and thyrotropin (TSH) show low-level cross-reactivity between their respective receptors (R). Patient serum autoantibodies to the thyrotropin receptor (TSHR) do not appear to cross-react with the luteinizing hormone receptor (LHR) or follicle-stimulating hormone receptor (FSHR), although the concentrations of autoantibody with which it is feasible to carry out experiments of this type are limited. Consequently, we have studied the effects of high doses of the thyroid-stimulating human monoclonal autoantibody (M22) on the LHR and FSHR. DESIGN: Chinese Hamster ovary (CHO) cells stably expressing the TSHR, LHR, and FSHR and purified M22 IgG preparations were used in the study. METHODS: CHO-TSHR, CHO-LHR, and CHO-FSHR cells were incubated with bovine TSH (0.1-25mU/mL), human recombinant chorionic gonadotropin (hCG; 0.5-10mU/mL) or human recombinant FSH (100-5000mU/mL) or with M22 IgG (0.001-5.0 microg/mL), and the extracellular cyclic AMP was measured by radioimmunoassay. RESULTS: Cyclic AMP levels increased in a dose-dependent manner after incubation of CHO-TSHR cells with TSH or M22 IgG, and on a molar basis the effects of TSH and M22 were similar. Cyclic AMP stimulation was not detectable in CHO-LHR and CHO-FSHR cells after incubation with M22 IgG, whereas incubation with hCG or FSH, respectively, caused dose-dependent cyclic AMP stimulation. On a molar basis, concentrations of M22 IgG approximately 100x those of FSH causing clear stimulation were ineffective with CHO-FSHR cells. Similarly, molar concentration of M22 IgG 20,000x those of hCG causing clear stimulation had no effect on CHO-LHR cells. CONCLUSIONS: This study shows that at relatively high concentrations, M22 IgG is unable to stimulate cyclic AMP levels in CHO-LHR or CHO-FSHR cells, suggesting that TSHR autoantibodies have greater specificity for the TSHR than TSH itself.

The sodium-iodide symporter expression in placental tissue at different gestational age: an immunohistochemical study.

BACKGROUND: Iodide (I(-)) is crucial for foetal thyroid function. Foetal iodide results from maternal circulating iodide and from deiodination of iodothyronines within the placenta. The Na(+)/I(-) symporter (NIS) localized in placental cells appears to be involved in iodide exchange. Low NIS expression has been reported in trophoblast cells from the first trimester and pregnancy at term. AIMS: The aim of this study was to examine NIS expression by immunohistochemistry in the major components of human ovular tissue and placenta. MATERIALS AND METHODS: Formalin-fixed and paraffin-embedded specimens of placental tissue from the first trimester and at term were analysed. NIS expression was quantified as percentage of NIS-positive cells/total cells. NIS expression was also evaluated by real-time polymerase chain reaction (RT-PCR) in five first-trimester and five at-term placental specimens. RESULTS: In the first-trimester specimens heterogeneous NIS immunoreactivity was found in cyto-syncytiotrophoblast cells, with a range of NIS-positive cells from 5% to 80% (mean +/- SD 21.85 +/- 23.95), in mesenchymal and endothelial cells from 1% to 40% (14.5 +/- 11.16), in decidual cells from 5% to 40% (10.38 +/- 11.98) and in endometrial glands from 3% to 40% (21.86 +/- 13.93). In specimens from placenta at term, NIS-positive cyto-syncytiotrophoblast cells were between 5% and 40% (mean 17.85 +/- 18.15), mesenchymal and endothelial cells between 1% and 40% (13.67 +/- 12.16), decidual tissue between 5% and 30% (16.43 +/- 9.08), and endometrial glands between 3% and 40% (16.67 +/- 15.27). No significant differences in NIS expression were observed between the first trimester and placenta at term. A similar level of mRNA expression for the NIS gene was obtained by RT-PCR both in ovular material of the first trimester and in placenta at term. CONCLUSIONS: We found NIS to be expressed in various placental and ovular components and its expression to remain constant during pregnancy.

Genetic analysis of the follicle stimulating hormone receptor gene in women with polycystic ovary syndrome.

This study was designed to assess the relationship between mutations in the FSH receptor (FSHr) gene and polycystic ovary syndrome (PCOS) in Italian women. The study population included 50 patients with PCOS and 50 age- and body mass index (BMI)-matched controls. A complete anthropometrical, hormonal and pelvic ultrasonographic evaluation was performed in all subjects. Genomic DNA was extracted from peripheral lymphocytes and then each exon of the FSHr gene was amplified by PCR. The mutation identified was cloned and the functional properties were studied after transient expression in COS-7 cells. Direct sequencing of exons 1-10 of the FSHr gene revealed the presence of a heterozygous AAT/ATT mutation affecting the isoleucine residue at position 411, which was replaced by an asparagine, in the second transmembrane segment (I411N). This mutation was only found in one woman with PCOS and not in her parents. This mutation was not present in 50 age and BMI controls and in another 150 women not affected by PCOS. The functional study after transient expression in COS-7 cells revealed that this I411N had similar functional characteristics with respect to the wild type FSHr (wtFSHr). Genetic analyses of polymorphisms in the human FSHr gene were also performed. All 50 women with PCOS harbored the A307T polymorphic variant, 56% harbored N680S, 30% S680S and 14% N680N polymorphisms. In conclusion, the present study demonstrates that mutations of the FSHr gene are rare in Italian women. The only mutation that we found does not appear to have any pathophysiological significance in PCOS.