Anti-thyroid hormonal activity of tetrabromobisphenol A, a flame retardant, and related compounds: Affinity to the mammalian thyroid hormone receptor, and effect on tadpole metamorphosis

The thyroid hormone-disrupting activity of tetrabromobisphenol A (TBBPA), a flame retardant, and related compounds was examined. TBBPA, tetrachlorobisphenol A (TCBPA), tetramethylbisphenol A (TMBPA) and 3,3'-dimethylbisphenol A (DMBPA) markedly inhibited the binding of triiodothyronine (T3; 1 x 10(-10) M) to thyroid hormone receptor in the concentration range of 1 x 10(-7)-1 x 10(-4) M, while bisphenol A and 2,2-diphenylpropane were inactive. TBBPA, TCBPA, TMBPA and DMBPA did not exhibit thyroid hormonal activity in a thyroid hormone-responsive reporter assay using a Chinese hamster ovary cell line (CHO-K1) transfected with thyroid hormone receptor alpha1 or beta1, but TBBPA and TCBPA showed significant anti-thyroid hormone effects on the activity of T3 (1 x 10(-8) M) in the concentration range of 3 x 10(-6) - 5 x 10(-5) M. The thyroid hormone-disrupting activity of TBBPA was also examined in terms of the effect on amphibian metamorphosis stimulated by thyroid hormone. TBBPA in the concentration range of 1 x 10(-8) to 1 x 10(-6) M showed suppressive action on T3 (5 x 10(-8) M)-enhancement of Rana rugosa tadpole tail shortening. These facts suggest that TBBPA, TCBPA, TMBPA and DMBPA can act as thyroid hormone-disrupting agents.     

Suppressing effects of bisphenol A on the secretory function of ovine anterior pituitary cells

We investigated the action of bisphenol A (BPA) on cellular GH release and content, cell number, GHmRNA expression, and concentrations of cellular cyclic AMP ([cAMP]c) and calcium ion ([Ca2+]c) in primary cultured ovine anterior pituitary cells. The following results were found: (1) BPA as well as nonylphenol (NP) at 10(-6) to 10(-3) M significantly and concentration-dependently suppressed basal and GHRH-stimulated GH release, and the cellular GH content, (2) BPA suppressed the cell number in a time- and concentration-dependent manner, (3) 10(-4)M BPA suppressed GHmRNA expression to 68% of control (BPA-free), and abolished GHRH (10(-8) M)-induced increases in [cAMP]c and [Ca2+]c. From these findings we conclude that BPA possesses a suppressing action on GH synthesis and release, and this suppressing action is probably related to impairment of cellular signal transduction systems in ovine anterior pituitary cells.     

In vitro pituitary hormone releasing activity of 40 residue human pancreatic tumor growth hormone releasing factor

The hypophysiotropic activities of a synthetic human pancreatic growth hormone releasing factor (hpGRF) with 40 residues was examined in vitro using rat pituitary halves. At concentrations from 10(-10) M to 10(-7) M the peptide stimulated GH release in a dose-dependent manner with the ED50 being 1.2 x 10(-9) M. The concentration of 10(-10) M hpGRF is comparable to the basal hypophyseal portal blood levels of other known hypothalamic hypophysiotropic hormones. However, GH release was enhanced three-fold by concentration as low as 10(-12) M, though no dose-response relationship was observed up to 10(-10) M. Thus, this peptide not only stimulates the release of GH in a dose-dependent manner, but at lower concentrations also maintains elevated GH levels. The release of ACTH, beta-endorphin, LH, and FSH was not affected by hpGRF at any of the concentrations tested. At hpGRF concentrations less than 10(-7) M, the release of TSH and PRL were unaffected. However, at 10(-6) M, TSH release was enhanced about 2.5 fold and prolactin release was elevated slightly.     

Ontogeny of the response to growth hormone-releasing factor

Primary cell cultures were prepared from fetal, neonatal and adult rat pituitaries and evaluated for their ability to secrete growth hormone (GH) in response to growth hormone-releasing factor (GRF). Pituitary cells prepared from fetuses at days 19 and 21 of gestation, neonatal animals at the day of birth (day 0) or the following day (day 1) and peripubertal male rats showed full dose response curves to GRF with maximal GH release when stimulated with 1 X 10(-10) M rat GRF. At this concentration of GRF, the amount of GH released was not different from that elicited by activation of adenylate cyclase with 1 X 10(-5) M forskolin. In contradistinction, a preparation of cells from fetuses at day 18 of gestation did not show the same release of GH when challenged with 1 X 10(-10) M GRF and forskolin (0.057 +/- 0.001, compared to 0.076 +/- 0.003 micrograms/10(5) cells per 4.5 h), although the cells clearly responded to both secretagogues (basal levels of GH, 0.029 +/- 0.002 micrograms/10(5) cells per 4.5 h). While cells prepared from fetuses at day 21 of gestation or from animals after birth released 5-10% of their total cellular GH content, those prepared from 18- and 19-day fetuses released as much as 40% of their total GH suggesting there is a maturation of intracellular GH processing that occurs late in gestation. The results show that, in late pregnancy, the rat fetal pituitary is highly responsive to growth hormone-releasing factor and suggest that this peptide participates in regulating GH levels during the perinatal period.     

Epidermal growth factor and thyrotropin-releasing hormone act similarly on a clonal pituitary cell strain. Modulation of hormone production and inhbition of cell proliferation

GH(4)C(1) cells are a clonal strain of rat pituitary cells that synthesize and secrete prolactin and growth hormone. Chronic treatment (longer than 24 h) of GH(4)C(1) cells with epidermal growth factor (EGF) (10(-8) M) decreased by 30-40 percent both the rate of cell proliferation and the plateau density reached by cultures. Inhibition of cell proliferation was accompanied by a change in cellular morphology from a spherical appearance to an elongated flattened shape and by a 40-60 percent increase in cell volume. These actions of EGF were qualitatively similar to those of the hypothalamic tripeptide thyrotropin-releasing hormone (TRH) (10(-7) M) which decreased the rate of cell proliferation by 10-20 percent and caused a 15 percent increase in cell volume. The presence of supramaximal concentrations of both EGF (10(-8)M) and TRH (10(-7)M) resulted in greater effects on cell volume and cell multiplication than either peptide alone. EGF also altered hormone production by GH(4)C(1) cells in the same manner as TRH. Treatment of cultures with 10(-8) M EGF for 2-6 d increased prolactin synthesis five- to ninefold compared to a two- to threefold stimulation by 10(-7) M TRH. Growth hormone production by the same cultures was inhibited 40 percent by EGF and 15 percent by TRH. The half- maximal effect of EGF to increase prolactin synthesis, decrease growth hormone production, and inhibit cell proliferation occurred at a concentration of 5 x 10 (-11) M. Insulin and multiplication stimulating activity, two other growth factors tested, did not alter cell proliferation, cell morphology, or hormone production by GH(4)C(1) cells, indicating the specificity of the EGF effect. Fibroblast growth factor, however, had effects similar to those of EGF and TRH. Of five pituitary cell strains tested, all but one responded to chronic EGF treatment with specifically altered hormone production. Acute chronic EGF treatment with specifically altered hormone production. Acute treatment (30 min) of GH(4)C(1) cells with 10(-8) M EGF caused a 30 percent enhancement of prolactin release compared to a greater than twofold increase caused by 10(-7) M TRH. Therefore, although EGF and TRH have qualitatively similar effects on GH(4)C(1) cells, their powers to affect hormone release acutely or hormone synthesis and cell proliferation chronically are distinct.     

Effects of bisphenol A on thyroid hormone-dependent up-regulation of thyroid hormone receptor alpha and beta and down-regulation of retinoid X receptor gamma in Xenopus tail culture

We investigated effects of different concentrations (10(-7) - 10(-5) M) of bisphenol A (BPA), which is known as an estrogenic and anti-thyroid hormonal endocrine disrupter, on the expression of thyroid hormone receptor (TR) alpha and beta and retinoid X receptor (RXR) gamma mRNA in tails of stage 52-54 Xenopus tadpoles in organ culture in the presence or absence of different concentrations of triiodo-thyronine (T(3)). In the absence of T(3), BPA at any concentration examined did not show remarkable effects on tail length but blocked 10(-7) M T(3)-induced tail resorption in a concentration-dependent manner. Semi-quantitative analyses of TRalpha and TRbeta mRNAs by RT-PCR in the tail specimens indicated that BPA shows an apparent antagonistic effect towards the receptors and reduced their mRNA levels relative to controls. When administered together with 10(-7) M T(3), the antagonistic effects of BPA were detected more clearly and dose-dependently. While BPA prevented the autoinduction of both TRalpha and TRbeta genes by T(3), the effect was less marked on TRalpha than on TRbeta. BPA also moderately suppressed RXRgamma gene expression. Gene expression of RXRgamma, a partner for heterodimer formation of TRs, was supressed by T(3) alone and also by BPA alone, but no additive effects were observed so far as studied. The present study indicates that a relatively low concentration of BPA, 10(-7) M, as compared with those examined previously (10(-5) to 10(-4) M) by us and other investigators, acts as an antagonist of T(3) through suppression of TRalpha and TRbeta gene expression in Xenopus tail in culture.     

Mutagenicity of bisphenol A and its suppression by interferon-alpha in human RSa cells

Bisphenol A is used as a monomer in the production of polycarbonate plastic products. The widespread use of bisphenol A has raised concerns about its effects in humans. Since there is little information on the mutagenic potential of the chemical, the mutagenicity of bisphenol A was tested using human RSa cells, which has been utilized for identification of novel mutagens. In genomic DNA from cells treated with bisphenol A at concentrations ranging from 1x10(-7) to 1x10(-5)M, base substitution mutations at K-ras codon 12 were detected using PCR and differential dot-blot hybridization with mutant probes. Mutations were also detected using the method of peptide nucleic acid (PNA)-mediated PCR clamping. The latter method enabled us to detect the mutation in bisphenol A-treated cells at a dose (1x10(-8)M) equivalent to that typically found in the environment. Induction of ouabain-resistant (Oua(R)) phenotypic mutation was also found in cells treated with 1x10(-7) and 1x10(-5)M of bisphenol A. The induction of K-ras codon 12 mutations and Oua(R) mutations was suppressed by pretreating RSa cells with human interferon (HuIFN)-alpha prior to bisphenol A treatment. The cells treated with bisphenol A at the concentration of 1x10(-6)M elicited unscheduled DNA synthesis (UDS). These findings suggested that bisphenol A has mutagenicity in RSa cells as well as mutagens that have been tested in these cells, and furthermore, that a combination of the PNA-mediated PCR clamping method with the human RSa cell line may be used as an assay system for screening the mutagenic chemicals at very low doses.     

A permissive role for extracellular Ca2+ in regulation of prolactin production by 1,25-dihydroxyvitamin D3 in GH3 pituitary cells

A clonal strain of rat pituitary tumor cells (GH3) that spontaneously synthesizes and secretes prolactin (PRL) and growth hormone (GH) was used as model system to study the mechanism of action of 1,25-(OH)2D3. We have previously demonstrated that these cells possess specific cytosol binding proteins for 1,25-(OH)2D3 (Haug and Gautvik, 1985). When the GH3 cells were incubated in a serum-free, chemically defined medium of low extracellular Ca2+ concentration, 1,25-(OH)2D3 stimulated PRL production in a dose-dependent manner. The stimulation was detectable at 10(-11) M, and the maximum effect (2-fold increase) was observed at 10(-9) M (ED50 = 2 x 10(-11) M). The dose-response curve was bell-shaped, and at 10(-6) M 1,25-(OH)2D3 even suppressed PRL production to about 75% of controls. The stimulatory effect was first seen after 2 days and was maximal after 4 days. On a molar basis 25-OHD3 and 1-OHD3 were at least 100 times less potent than 1,25-(OH)2D3, while 24,25-(OH)2D3 had no effect on PRL production. At an extracellular concentration of Ca2+ as low as 4 x 10(-5) M the stimulatory effect of 1,25-(OH)2D3 was small (1.3-fold). Increasing extracellular Ca2+ to 1.5 x 10(-4) M increased the 1,25-(OH)2D3-induced PRL response to 2.1-fold. In contrast to the biphasic effect of 1,25-(OH)2D3 on PRL production, GH production was decreased to about 60% of controls at 10(-8) M and above. These findings indicate that in serum-free medium the stimulatory effect of 1,25-(OH)2D3 on PRL production is critically dependent on the concentration of extracellular Ca2+.     

Defective thyroliberin-induced prolactin synthesis and release in a hybrid GH strain

Summary The hybrid GH cell strain, 928-9b, isolated from PRL+ (prolactin [PRL] producing) GH₄Cl and PRL (PRL non-producing) FIBGH12CI cells, has specific TRH (thyroliberin) receptors, yet does not respond to this peptide hormone. Unlike the parent strain, GH₄Cl, TRH does not stimulate synthesis or release of PRL in the hybrid strain. In contrast, treatment of 928-9b cells with another peptide, EGF (epidermal growth factor), stimulates both release and synthesis of PRL. The number of EGF receptors in the hybrid strain (2.5 × 10³/cell) and the affinity of these receptors for ligand (2.2 nM) are comparable to that of the parent strain, GH₄C₁. The EGF dose response curve is also essentially the same for parent and hybrid cells for the enhancement of PRL production. A 3-8-fold enhancement of PRL production is observed and 1/2 maximal enhancement occurs at approximately 5 × 10¹¹ M EGF for both strains. TRH does not have any potentiating effect on EGF-induced stimulation of PRL release or PRL synthesis in the hybrid strain. Although EGF and TRH have similar biological effects in responsive GH cells, binding of one hormone to its receptors does not modulate the binding of the heterologous hormone. These findings demonstrate that more than one effect of TRH is defective in 928-9b cells even though EGF responses are intact. This suggests that 1) TRH-stimulated PRL release and TRH-stimulated PRL production have a common intermediate step, and 2) TRH and EGF have a different mechanism of action in GH cells.     

Differentiation between the somatostatin inhibition and the post-somatostatin rebound observed on growth hormone secretion in vitro

A rebound in growth hormone secretion following somatostatin treatment has been shown in several systems where somatostatin suppresses secretion of the hormone. We have developed an in vitro system in which isolated and cultured pituitary cells were perfused after mild trypsinization. After washing, these cells retained their sensitivity and secreted growth hormone (GH) in response to physiological activators (norepinephrine, dopamine, serotonin) or inhibitors (somatostatin) as well as pharmacological activators (PGE2). The variation in GH secretion occurred within a minute after commencement of the infusion and was as rapidly reversible and repeatable minutes later. During somatostatin infusion the GH secretion was not totally suppressed (residual secretion (mean +/- S.D.) 34 +/- 7%). After the infusion a rapid rebound in GH secretion occurred, reaching levels in excess of the pretreatment value of 138 +/- 13%. This rebound effect occurred at doses higher than (10(-10)M) but not at lower doses, even when significant inhibition was observed. The inhibitory effect is of greater magnitude than the rebound effect (rebound = inhibition X 57 +/- 7% (mean +/- S.D.)). Furthermore, rebound was not enhanced by prolongation of somatostatin infusion. These latter results indicate that the rebound in secretion cannot be explained on the sole basis of storage of intracellular GH during somatostatin infusion and in fact suggest the involvement of a process of GH degradation and/or an inhibition of GH synthesis.     

Characterization of functional receptors for somatostatin in rat pituitary cells in culture.

GH4C1 cells are a clonal strain of rat pituitary tumor cells which synthesize and secrete prolactin and growth hormone. Somatostatin, a hypothalamic tetradecapeptide, inhibits the release of growth hormone and, under certain circumstances, also prolactin from normal pituitary cells. We have prepared [125I-Tyr1]somatostatin (approximately 2200 C1/mmol) and have shown that this ligand binds to a limited number of high affinity sites on GH4C1 cells. Half-maximal binding of somatostatin occurred at a concentration of 6 x 10(-10) M. A maximum of 0.11 pmol of [125I-Tyr1]somatostatin was bound per mg of cell protein, equivalent to 13,000 receptor sites per cell. The rate constant for binding (kon) was 8 x 10(7) M(-1) min(-1). The rate constant for dissociation (koff) was determined by direct measurement to be 0.02 min(-1) both in the presence and absence of excess nonradioactive somatostatin. Binding of [125I-Tyr1]somatostatin was not inhibited by 10(-7) M thyrotropin-releasing hormones. Substance P, neurotensin, luteinizing hormone-releasing hormone, calcitonin, adrenocorticotropin, or insulin. Of seven nonpituitary cell lines tested, none had specific receptors for somatostatin. Somatostatin was shown to inhibit prolactin and growth hormone production by CH4C1 cells. The dose-response characteristics for binding and the biological actions of somatostatin were essentially coincident. Furthermore, among several clonal pituitary cell strains tested, only those which had receptors for somatostatin showed a biological response to the hormone. We conclude that the characterized somatostatin receptor is necessary for the biological actions of somatostatin on GH4C1 cells.     

Changing ultrastructure of thyrotrophs in the rat anterior pituitary after thyroidectomy as studied by immuno-electron microscopy and enzyme cytochemistry

Summary The characteristic ultrastructure of thyrotrophs of the rat anterior pituitary was observed by immuno-electron microscopy and enzyme cytochemistry with increasing time after thyroidectomy (TX). The rough endoplasmic reticulum (ER) became dilated, the intracisternal granules reacted to serum raised against thyroid stimulating hormone (TSH) around 21 days after TX, and lysosomes and peculiar structures with positive acid phosphatase activity were present. The administration of thyroxine (T₄) to the thyroidectomized rats resulted in the reformation of secretory granules, a reduction of dilated cisternae of rough ER and the activation of the lysosomal systems. Morphological features indicating that the TX-cells might be derived from growth hormone (GH) cells or cells other than TSH cells, previously suggested by some researchers, were not recognized in the present study. The amount of serum and pituitary TSH was measured by radioimmunoassay (RIA), and correlated well with the morphological changes. These results indicate that the TX-cells are hypertrophied hyperfunctioning TSH cells that have been affected by the lack of negative feedback of thyroid hormone.     

Influence of hormone concentration and time factor on development of receptor memory in a unicellular (Tetrahymena) model system

1. Treatment of Tetrahymena pyriformis cells with diiodotyrosine (T2) gave rise to a considerable, concentration-dependent increase of the growth rate within the range of 10(-15) and 10(-9) M, but did not influence it at the level of 10(-18) M. 2. Re-exposure of the cells 1, 2 and 4 weeks later to the hormone concentrations originally used accounted for a marked increase of growth rate at all hormone levels tested, indicating that the extremely low concentration of 10(-18) M, which failed to stimulate growth on first exposure, did nevertheless give rise to hormonal imprinting, which caused the cells to "remember" the hormone, as judged from their increased responsiveness to it on re-exposure. 3. The degree of growth response was concentration-dependent on both first and second exposure: higher levels of treatment gave rise to firmer imprinting, and to greater response on re-exposure. 4. The length of exposure time proved to be more decisive than the level of treatment in respect of the development of hormonal imprinting. 5. Short-term exposures up to 60 min, although they stimulated cell growth by direct effect, gave rise to lasting inhibition of cellular response to re-exposure(s) rather than to hormonal imprinting.     

A comparison of the biological activities of authentic rat GRF(1-43)OH with the analogue rat GRF(1-29)NH2

The purpose of this study was to characterize the biological activity of the synthetic rat growth hormone releasing factor analogue rGRF(1-29)NH2 and to compare its action on growth hormone (GH) release to that of authentic rGRF(1-43)OH. We first compared the concentration-response characteristics of the two peptides in static incubation, and then examined the reversibility and repeatability of the GH response in a perifusion system. Authentic rGRF(1-43)OH was significantly more potent in static incubation (EC50 = 3 x 10(-11) M) than the analogue (5 x 10(-11) M), whereas the reverse held true in perifusion. The shapes of the GH responses were similar for both peptides in the perifusion system. However, while the GH response to authentic rGRF was repeatable, the prior administration of rGRF(1-29)NH2 significantly reduced (greater than 50%) the GH response to the subsequent administration of either rGRF(1-29)NH2 or rGRF(1-43)OH. Thus authentic rGRF and the synthetic fragment may have different actions at the level of the GRF receptor or at a postreceptor (second messenger) step.     

Receptor for 1,25-dihydroxyvitamin D3 in GH3 pituitary cells

Cytosol prepared in 0.3 M KCl from pituitary GH3 cells, but not from AtT-20 cells contains a receptor-like macromolecule that binds 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) with specificity and high affinity (Kd = 2.9 x 10(-10) M). The GH3 cytosolic binding component sediments at 3.3 S in high-salt sucrose gradients and adsorbs to DNA-cellulose; its elution profile from DNA-cellulose and other biochemical properties are indistinguishable from those of classical 1,25(OH)2D3 hormone receptors. The presence of the 1,25(OH)2D3 receptor in pituitary cells which secrete primarily growth hormone and prolactin (GH3), but not in a line which secretes the 31,000-dalton ACTH precursor and its derived peptides (AtT-20), suggests that 1,25(OH)2D3 may play a regulatory role in specific pituitary cells.     

Fine-structural and immunohistochemical study of anterior pituitary cells of Snell dwarf mice

Summary Snell dwarf mice display remarkable retardation of growth after birth and are known to lack prolactin (PRL), thyroid stimulating hormone (TSH) and growth hormone (GH). The aim of this study was to determine the reason for these hormonal deficiencies. We examined the fine structure of the gland and its immunohistochemical staining pattern with respect to antisera raised against PRL, TSH, GH, adrenocorticotrophic hormone (ACTH) and luteinizing hormone (LH). The gland of control mice reacted immunohistochemically against all antisera used, whereas only ACTH-producing cells (ACTH cells) and LH-producing cells (LH cells) were distinguished in the dwarf mice. ACTH cells in dwarf mice varied in cell shape, although they were similar in size to those of controls. The distribution of secretory granules in the cytoplasm varied from cell to cell. LH cells in the dwarf mice showed immature features, having poorly developed rough endoplasmic reticulum and Golgi apparatus. The cells were about half the size of controls, and secretory granules were smaller. In dwarf mice, non-granulated cells were encountered in addition to granulated ACTH and LH cells. Some of them formed small clusters, characteristic cell junctions being found between the cells; they thus appeared to be follicular cells. The above results suggest that hormone deficiency in Snell dwarf mice is a result of a defect in the hormoneproducing cells in the gland.     

Differential regulation of thyrotropin-releasing hormone receptor mRNA levels by thyroid hormone in vivo and in vitro (GH3 cells).

We studied the effect of thyroid status on thyrotropin-releasing hormone receptor (TRH-R) mRNA levels both in vivo and in vitro (GH3 cells) using a cloned rat TRH-R cDNA by RT-PCR. Experimental hypothyroid rats were produced by total thyroidectomy and were then killed 7 days after the operation. TRH receptor binding in the anterior pituitary and serum TSH level were elevated approximately 2-fold and 8-fold, respectively, in 7 day thyroidectomized rats. TRH-R mRNA levels in hypothyroid rats were also increased significantly compared with those of normal rats. In GH3 cells, however, no significant change of TRH-R mRNA level was observed between cultures treated with triiodothyronine (T3, 10(-9) and 10(-7) M) and the untreated group. The present data indicate that 1) the in vivo effects of thyroid status on TRH-R mRNA levels differ from the in vitro one, and that 2) the down regulation of TRH-R binding by thyroid hormone in GH3 cells may be mediated by translational or post-translational mechanisms.     

瘦素对GH3细胞分泌和凋亡的影响

本文旨在探讨瘦素(leptin)对垂体瘤GH3细胞的生长激素(growth hormone,GH)分泌的作用及可能机制。我们观察了leptin对GH3细胞生长激素的分泌、细胞的增殖和凋亡的影响,结果显示:leptin(1、10和100 nmol/L)对GH3细胞的基础GH分泌有抑制作用(P<0.05),并存在剂量依赖效应。用10 nmol/L的leptin作用30 min、1和3 h对GH分泌无明显影响,而作用1、2和3 d则可抑制GH分泌(P<0.05)。应用噻唑蓝(MTT)比色分析法和流式细胞仪研究leptin对GH3细胞增殖和凋亡的影响,我们发现leptin对GH3细胞的增殖有抑制作用,并存在剂量依赖效应;同时leptin可减低GH3细胞的S期细胞比例,而G1期的细胞比例明显增加,进入2相和4相的凋亡细胞比例增加。上述结果表明,leptin可抑制GH3 细胞的基础GH分泌,其作用可能是通过抑制GH3细胞的DNA合成,促进GH3细胞的凋亡,从而影响GH的分泌。