Claudia Mammoli

In vitro assay of thyroid disruptors affecting TSH-stimulated adenylate cyclase activity

Several natural or synthetic chemicals have been indicated as potential thyroid disruptors. The development of in vitro assays has been recommended to comprehensively assess the potential thyroid disrupting activity of a substance or a complex mixture. In this study, 12 substances suspected for acting as thyroid disruptors were tested for their ability to inhibit TSH-stimulated cAMP production in vitro. Those substances producing an inhibition were further studied to establish the level at which they interfere with this step of thyroid cell function. Using Chinese hamster ovary cells (CHO) transfected with the recombinant human TSH receptor, a dose-dependent inhibition of TSH-stimulated adenylate cyclase activity was produced by 1,1-bis-(4-chlorphenyl)-2,2,2-trichloroethan (DDT), Aroclor 1254 and Melissa Officinalis. All three substances also inhibited the cAMP production stimulated by TSH receptor antibody. Melissa Officinalis produced a significant inhibition of TSH binding to its receptor and of antibody binding to TSH, while no significant changes were produced by Aroclor 1254 or DDT in these assays. These data suggest that principles contained in Melissa Officinalis may block the binding of TSH to its receptor by acting both on the hormone and the receptor itself, while DDT and Aroclor 1254 affect cAMP production mainly at post-receptor step. In conclusion, we have developed a set of in vitro assays that allow investigation into the effect of thyroid disruptors on the TSH-mediated activation of the cAMP cascade. These assays may be useful to identify the mechanism of action of thyroid disruptors, coming beside and supporting animal studies or epidemiological surveys.

Serum concentrations of adiponectin and leptin in patients with thyroid dysfunctions.

Thyroid dysfunction is associated with metabolic changes that affect mass and adipocyte function, as well as lipid and carbohydrate metabolism. Adipose tissue performs complex metabolic and endocrine functions. Leptin and adiponectin are two of the most important adipocytokines, both involved in the regulation of intermediate metabolism. The aim of this study was to evaluate the relationships between thyroid status and circulating levels of the two adipose tissue hormones. We studied 15 patients with hyperthyroidism, 15 patients with hypothyroidism and 15 euthyroid subjects, all matched by sex, age and body mass index (BMI). Serum concentrations of free thyroxine, free triiodothyronine, thyrotropin, leptin and adiponectin and anthropometric parameters (weight, height, BMI) were assessed. No significant difference was found among the 3 groups, as assessed by Student’s t-test, both for adiponectin and leptin. We conclude that metabolic changes associated with thyroid dysfunction are not related to variations in serum levels of adiponectin or leptin.

HUMAN SERUM THYROTROPHIN MEASUREMENT BY ULTRASENSITIVE IMMUNORADIOMETRIC ASSAY AS A FIRST-LINE TEST IN THE EVALUATION OF THYROID FUNCTION

An ultrasensitive immunoradiometric assay (IRMA) using two monoclonal anti‐TSH antibodies has been used for TSH measurements in basal conditions and after TRH stimulation. The results have been compared with those obtained by conventional radioimmunoassay (RIA). The IRMA method had very high sensitivity (0·07 μU/ml). Detectable serum TSH concentrations were found in all normal subjects by IRMA, but in only 76% by RIA. No overlap was observed with the results obtained by IRMA in untreated overtly hyperthyroid patients, in whom serum TSH was below the limit of detection. The relationship between basal and TRH‐stimulated serum TSH concentrations by IRMA and RIA was evaluated in 176 subjects including normals and patients with untreated and treated hyperthyroidism, functioning thyroid adenoma, non‐toxic goitre and patients on l‐thyroxine therapy. A normal TSH response to TRH was observed in virtually all patients with detectable basal serum TSH by both methods. When patients with undetectable basal serum TSH levels were considered, all but one (98%) had no TSH response to TRH by IRMA. On the contrary using RIA, an absent response was found only in 47% of subjects, a blunted responses in 10% and a normal response in 42%. These data indicate that basal serum TSH measurements by IRMA allows a complete discrimination of normal from hyperthyroid patients and can avoid the need for TRH stimulation tests.

Measurement of cAMP accumulation in chinese hamster ovary cells transfected with the recombinant human TSH receptor (CHO-R) : a new bioassay for human thyrotropin

Circulating TSH bioactivity may vary in several clinical and experimental conditions. Since the reliability of the current methods for the measurement of TSH bioactivity is limited, a new bioassay based on cAMP accumulation in Chinese Hamster Ovary cells transfected with recombinant human TSH receptor (CHO-R) was set up. The sensitivity was 0.3±0.1, 0.4±0.1 and 0.01±0.01 μg/L for TSH IRP 80/558, recombinant human TSH and bovine TSH, respectively. Standard curves were parallel, and the intra- and inter-assay coefficients of variation were 13±1.1% and 22±1.9%, respectively. LH, FSH, CG and TSH subunits did not stimulate cAMP accumulation up to high concentrations. Circulating TSH was partially purified by immunoaffinity separation and concentrated before being bioassayed. However, plain sera with high TSH levels, such as those from primary hypothyroid patients (PH), could be directly tested in CHO-R bioassay, provided that sera were added at concentrations lower than 10%. TSH from 6 normal subjects had biological to immunological ratio (B/l) ranging from 0.6 to 2.1 (mean±SD=1.4±0.5). TSH from 6 patients with PH showed bioactivity significantly lower than in normals (B/l=0.6±0.3; p<0.001; range=0.3–1.1). TSH from 5 patients with central hypothyroidism of hypothalamic origin (CH) had undetectable basal bioactivity (B/l <0.2), which normalized in only one patient after acute TRH and in all patients after chronic TRH administration. In conclusion, CHO-R cells provide an excellent tool for evaluating TSH bioactivity, owing to high sensitivity, specificity, reproducibility and feasibility of the assay. Our findings demonstrate that TSH B/l in patients with PH is significantly lower than in normal subjects and confirm that TSH molecules secreted in patients with CH have undetectable bioactivity which is normalized by TRH treatment.

Serum thyrotropin by ultrasensitive immunoradiometric assay and serum free thyroid hormones in pregnancy

Variations of serum TSH, measured by an ultrasensitive immunoradiometric assay, of serum total and free thyroid hormones and of thyroxine-binding globulin (TBG) and sex hormone-binding globulin (SHBG) were investigated in a group of 18 normal women before and during pregnancy. A gradual increase of total thyroid hormones, TBG and SHBG was observed, while mean serum free thyroxine and free triiodothyronine progressively decreased. Serum TSH concentrations were comprised within the normal range throughout pregnancy, although a small but significant increase was found in the 2nd and 3rd trimester. These changes may represent a compensatory mechanism to meet the increased demand for thyroid hormones in pregnancy and must be taken into account for a correct evaluation of thyroid function during gestation.

Detection of antibodies blocking thyrotropin effect using Chinese hamster ovary cells transfected with the cloned human TSH receptor

Chinese hamster ovary (CHO) cells transfected with the cloned human TSH receptor (CHO-R) were used to develop an assay to detect thyroid autoantibodies blocking the TSH-dependent cAMP production (TSHBAb). The study group included 38 patients with goitrous Hashimoto’s thyroiditis (HT) and 47 subjects with atrophic thyroiditis (AT). In the HT group, 8 patients had subclinical hypothyroidism (HT-SH) and 30 had overt hypothyroidism (HT-H). Thirty normal subjects served as controls. Immunoglobulin G (IgG) was prepared from serum by double chromatography on DEAE-Sephadex. CHO-R cells were seeded in 96-well plates and were cultured for 48 h before the assay in RPMI-1640 medium plus 1 mmol/L glutamine, 10% fetal calf serum, and 0.4 g/L geneticin. In the assay for TSHBAb, CHO-R cells were incubated with IgG alone (0.5–2 mg/ml), TSH alone (0.2–625 mU/L), or IgG plus TSH; all samples were diluted in hypotonic medium containing 0.5 mmol/L isobutylmethylxanthine (IBMX). After 2 h of incubation at 37°C in 5% CO2 −95% air atmosphere, TSH-stimulation was quantified by measuring extracellular cAMP by a RIA. IgGs from normal subjects did not significantly modify the stimulation of adenylate cyclase produced by TSH, the results obtained ranging between −30% and +18% (mean±SD=−3±14%). All IgGs producing an inhibition greater than 2SD from the mean of controls (>25%) were considered positive for blocking antibodies. TSHBAb were detected in 1/8 (12.5%) patients with HT-SH, in 7/30 (23.3%) with HT-H and in 16/47 (34.0%) patients with AT. When the same IgGs were tested in FRTL-5 cells, TSHBAb were detected in 1/8 (12.5%) patients with HT-SH, in 5/30 (16.6%) with HT-H and in 15/47 (31.9%) with AT. TSHBAb results in CHO-R cells showed a good correlation with those in FRTL-5 cells (r=0.74, p<0.0001), but 3/24 IgGs were positive for TSHBAb in CHO-R cells and negative in FRTL-5 cells. Using the radioreceptor assay, TSH-binding inhibiting antibodies were detected in 17/24 (70.8%) sera that contained TSHBAb when tested in the CHO-R cell system. Thyroid stimulating antibody (TSAb) and TSHBAb, that coexisted in 5 IgGs, were simultaneously detected using CHO-R cells. These IgGs belonged to patients in whom spontaneous hypothyroidism developed after hyperthyroidism, or viceversa. In conclusion a new in vitro assay for the detection of TSHBAb was developed using CHO-R cells. The sensitivity of this assay is slightly greater than that obtained in FRTL-5 cells and definitely greater than that of the radioreceptor assay. CHO-R cells have the advantages of expressing the human TSH receptor and of requiring less cumbersome procedures for cell culture than FRTL-5 cells.

Favorable predictive value of thyroid autoimmunity in high aggressive breast cancer

A high incidence of anti-thyroid antibodies (TAb) has been found in patients with breast cancer (BC). The aim of this study was to evaluate the prognostic value of TAb in a group of 47 women submitted to mastectomy for high malignancy degree BC. All patients were evaluated for thyroid disorders after breast surgery and before any anti-tumoral adjuvant therapy. Five yr after BC diagnosis 31/47 (65.9%) patients were alive (survivors group: SG) and 16/47 (34.1%) were dead (deaths group: DG). The overall prevalence of TAb was 15/47 (31.9%): 14/31 (45.1%) in SG and 1/16 (6.2%) in DG (p=0.008). Five-yr mortality was 15/32 (46.9%) in TAb- and 1/15 (6.7%) in TAb+ patients (p=0.01). Eight out of 47 (17.0%) patients had Hashimoto’s thyroiditis and 7 of them (87.5%) were in SG. Estrogen receptor (ER) was measured in 43/47 (91.5%) BC specimens. ER was detected in 19/30 (63.0%) patients in SG and 3/13 (23.1%) in DG (p=0.01). Five-yr mortality was 10/21 (47.6%) in ER- and 3/22 (13.6%) in ER+ patients (p=0.008). Absence of ER expression [odds ratio (OR) 6.54; p=0.006] and absence of TAb (OR 9.37; p=0.03) were related to a higher mortality rate. TAb were detected in 8/21 (38.1%) ER- and in 7/22 (31.8%) ER+ patients; no relation was found between ER expression and TAb positivity (p=ns). Patients with ER+ and TAb+ have a better prognosis and the absence of a significant relationship between these two parameters suggests an independent prognostic role in high malignancy degree BC women.

Detection and characterization of autoantibodies blocking the TSH-dependent cAMP production using FRTL-5 cells

Autoantibodies blocking the TSH-stimulated cAMP production (TBkAb) were measured in immunoglobulin G (IgG) preparations from 38 patients with primary autoimmune hypothyroidism, using FRTL-5 cells. TBkAb were detectable in 15/23 IgG preparations from patients with untreated idiopathic myxedema, and in 2/15 IgGs from patients under L-thyroxine treatment. None of the IgG from 22 normal subjects or from 10 patients with nonautoimmune hypothyroidism following total thyroidectomy caused any significant effect on the TSH-stimulated cAMP production. No correlation was found between TBkAb and the thyroid microsomal antibody. Antibodies inhibiting the 125I-TSH binding to TSH receptor were detectable in only 3/20 patients; IgGs from these 3 patients were also positive in the TBkAb assay. One IgG with potent TBkAb activity inhibited the TSH-stimulated adenylate cyclase in a competitive manner, while it had no effect on the forskolin-stimulated cAMP production. The inhibiting action of this IgG was almost completely lost after preabsorption with human thyroid membranes. In conclusion, we describe a new practical and sensitive method for the measurement of TBkAb; TBkAb are distinct from the microsomal antibody, and are probably directed to the TSH receptor.

Evaluation of L-thyroxine replacement therapy in children with congenital hypothyroidism

The outcome of L-thyroxine (L-T4) replacement therapy in children with congenital hypothyroidism (CH) remains to be completely evaluated. In this paper the overall pattern of response to L-T4 replacement therapy was studied in a group of 19 children with CH diagnosed by neonatal screening (10 with hypoplastic/aplastic thyroid disease, group H/A; 9 with gland ectopy, group E) who were followed-up for 60 ± 27 months (mean ± SD). With 1 exception serum T4 at diagnosis was > 2 µg/dl in children of group E and < 2 µg/dl in those of group H/A. The initial dose of L-T4 (8–10 µg/kg BW/day) was modified in relation to age and weight in order to maintain serum TSH ≼ 5 µU/ml and FT3 in the normal range. A general inverse correlation between serum TSH and FT4 or FT3 concentrations was found, and the mean levels of serum FT4 and FT3 were significantly higher according to the following order of TSH results: low TSH (0–0.5 µU/ml) > normal (>0.5–5 µU/ml) > elevated TSH (>5 µU/ml). TSH levels < 5 µU/ml were associated with FT4 values in the upper half of the normal range (54% of observations) or even higher (46%). Elevation of serum FT4 alone with FT3 values in the normal range did not result in clinical thyrotoxicosis, alteration of growth or premature craniosynostosis. Mean L-T4 doses (µg/kg BW/day) administered to CH children were: 7.0 ± 1.6 between 1 and 6 months; 5.2 ± 0.9 between 6 and 12 months; 4.0 ± 0.6 between 1 e 5 yr; 3.4 ± 0.6 over 6 yr. In general, the mean L-T4 closes given to children showing low, normal or elevated TSH were widely overlaped. Growth in weight and height was normal, no significant difference being observed in children of group H/A vs. those of group E. Mental development was within the normal range, but at age 12 months children in group H/A had significantly lower DQ scores than those in group E, and at age 3 yr both groups of CH children had significantly lower IQ scores than unaffected controls. In conclusion: i) in children with CH serum TSH can be suppressed to normal or even subnormal concentrations provided that enough L-T4 is given to maintain FT4 in the upper half of the normal range or slightly higher; ii) sporadic elevations of TSH during treatment could be attributed to: failure to reassess the dose of L-T4 following the infant’s rapid gain in weight, lack of compliance, malabsorption of the drug, misunderstanding of prescription; iii) neuropsychological development was within the normal range of tests; however, the absence of functioning thyroid tissue, as shown by low T4 at diagnosis and by negative radioisotope imaging, was a risk factor for lower mental scores.

Measurement of TSAb directly in serum using FRTL-5 cells

FRTL-5 cells were shown to be suitable for the measurement of thyroid stimulating antibody (TSAb) present in sera of patients with Graves’ disease. Current methods for the assay of TSAb require the separation of immunoglobulin G (IgG)from patient sera. In this report the possibility to measure TSAb directly on serum was evaluated using FRTL-5 cells. To this purpose cells were seeded in 96-well plates and cultured for 4 days in medium deprived of TSH. Using this system bovine TSH was able to produce a significant stimulation of cAMP production at 1 μU/ml. Whole normal serum completely inhibited the stimulation of TSH as well as that of TSAb, while diluted serum was devoid of any effect. Heat inactivated sera and IgGs, prepared by DEAE Sephadex separation, were diluted in hypotonic medium and incubated with cells for 1 h at 37 C. After incubation cAMP was measured in the assay medium by RIA. In some experiments the effects of graded dilutions of sera and IgGs with known TSAb activity were compared. Sera as well as IgGs increased the cAMP production, but, at the highest concentrations, an inhibitory effect was evident. For this reason sera were tested after appropriate dilution. Thirteen/27 (48%) sera and 22/27 (81%) IgGs from patients with Graves’ disease were TSAb positive. The effect of Graves’ sera on adenylate cyclase stimulation was completely inhibited by an anti-human IgG. The results of stimulation produced by Graves’ sera and IgGs were highly correlated (r = 0.97, p < 0.001 ). In conclusion it is possible to measure TSAb directly in serum using FRTL-5 cells. This assay is less sensitive then the assay that employs the purified IgG. However, positive results were found in almost 50% of unselected Graves’ patients. Thus, it is reasonable to propose this assay as a first-line screening for TSAb; the assay employing purified IgGs can be limited to negative sera.