Thyroid hormonal activity of the flame retardants tetrabromobisphenol A and tetrachlorobisphenol A

The thyroid hormonal-disrupting activity of the flame retardants tetrabromobisphenol A (TBBPA) and tetrachlorobisphenol A (TCBPA) was examined and compared with that of bisphenol A, a typical estrogenic xenobiotic. TBBPA and TCBPA, halogenated derivatives of bisphenol A, markedly inhibited the binding of triiodothyronine (T(3); 1 x 10(-10) M) to thyroid hormone receptor in the concentration range of 1 x 10(-6) to 1 x 10(-4) M, but bisphenol A did not. The thyroid hormonal activity of TBBPA and TCBPA was also examined using rat pituitary cell line GH3 cells, which grow and release growth hormone (GH) depending on thyroid hormone. TBBPA and TCBPA enhanced the proliferation of GH3 cells and stimulated their production of GH in the concentration range of 1 x 10(-6) to 1 x 10(-4) M, while bisphenol A was inactive. TBBPA, TCBPA, and bisphenol A did not show antagonistic action, i.e., these compounds did not inhibit the hormonal activity of T(3) to induce growth and GH production of GH3 cells. TBBPA and TCBPA, as well as bisphenol A, enhanced the proliferation of MtT/E-2 cells, whose growth is estrogen-dependent. These results suggest that TBBPA and TCBPA act as thyroid hormone agonists, as well as estrogens.     

The interaction of the brominated flame retardant: tetrabromobisphenol A with phospholipid membranes

Tetrabromobisphenol A (TBBPA) is one of the most widely used members of the family of brominated flame retardants (BFRs). BFRs, including TBBPA have been shown to be widely distributed within the environment and there is growing evidence of their bio-accumulation within animals and man. Toxicological studies have shown that TBBPA can be harmful to cells by modulating a number of cell signalling processes. In this study, we employed fluorescence spectroscopy and differential scanning calorimetry to investigate the interaction of TBBPA with phospholipid membranes, as this is the most likely route for it to influence membrane-associated cellular processes. TBBPA readily and randomly partitions throughout all regions of the phospholipid bilayer with high efficacy [partition coefficient (Log K(p))=5.7+/-0.7]. A decrease in membrane fluidity in both liquid-crystalline and gel-phase membranes was detected at concentrations of TBBPA as low as 2.5 microM. TBBPA also decreases the phase transition temperature of dipalmitoyl phoshatidylcholine (DPPC) membranes and broadened transition peaks, in a fashion similar to that for cholesterol. TBBPA, however, also prefers to partition into membrane regions not too highly enriched with cholesterol. Our findings therefore suggests that, the toxic effects of TBBPA, may at least in part, be due to its lipid membrane binding/perturbing effects, which in turn, could influence biological processes involving cell membranes.     

The vitamin D receptor: a primitive steroid receptor related to thyroid hormone receptor

We have previously reported the cloning and sequencing of both the chicken and human vitamin D3 receptor cDNAs. A comparison of their deduced amino acid sequence with that of the other classic steroid hormone receptors and the receptor for thyroid hormone indicates that there are two regions of conservation between these molecules. The first is a 70 amino acid, cysteine-rich sequence (C1), the second region (C2) is a 62 amino acid region located towards the carboxyl terminus of the proteins. In other systems the former has been identified as a region responsible for DNA binding activity, whereas the latter represents the NH2-terminal boundary of the hormone binding domain. We present here evidence utilizing eucaryotic expression of cDNA encoding the hVDR C1 domain, followed by a DNA cellulose chromatography assay, which confirms that the DNA binding activity resides in this region of the receptor for vitamin D3. Additionally, the vitamin D3 receptor contains a 60 amino acid portion at its carboxyl terminus (C3) which exhibits homology with the receptor for thyroid hormone. Conservation in this region of the molecule is found only between homologous or closely related receptors. This indicates a relationship between the vitamin D3 receptor and the receptor for thyroid hormone and may suggest that they evolved from a single primordial gene.     

Identification of nuclear factors that enhance binding of the thyroid hormone receptor to a thyroid hormone response element

Using a gel shift assay, we analyzed the binding of in vitro translated alpha- and beta-thyroid hormone (T3) receptors to a T3-response element (TRE) derived from the rat GH gene. No receptor-TRE complexes were observed when translated receptor alone was incubated with the TRE. However, addition of a nuclear extract from liver to the translational products resulted in the formation of two receptor-DNA complexes for both the alpha- and beta-receptors. These complexes were shown to contain translated receptor by comigration of 32P-labeled TRE and 35S-labeled receptor in the gel shift assay. A competition experiment demonstrated that formation of the complexes was sequence specific. Preincubation of the liver nuclear extract at 60 C abolished formation of both complexes indicating that receptor-TRE binding required a heat-labile nuclear factor. Phosphocellulose chromatography of the nuclear extract resulted in separation of the activities required for formation of the two complexes. Analysis of nuclear extracts from different tissues revealed that one complex formed in the presence of all extracts, whereas the second complex appeared predominantly with a nuclear extract from liver. Addition of T3 to the binding reaction had no effect on receptor-TRE complex formation. We suggest that nuclear factors interact with the T3 receptor to enhance hormone-independent binding to a TRE.     

Effect of thyroid hormone analogues on the displacement of 125I-L-triiodothyronine from hepatic and heart nuclei in vivo: possible relationship to hormonal activity

The capacity of iodotyrosines and iodothyronine analogues to displace tracer[¹²⁵I] L-3,5,3′ triiodothyronine from specific nuclear binding sites in rat liver and heart was related to the displacement capacity of nonradioactive triiodothyronine. Iodotyrosines and L-3,3′,5′ triiodothyronine (“reverse T₃”) were devoid of displacement activity. Analogues with 3,5 substitution in the “inner” ring and single “bulk” substitution in the 3′ position in the phenolic ring exhibited the strongest displacement activity. When the distribution, fractional removal rates and metabolic conversion of the analogues were taken into account, displacement activity appeared to correlate well with the reported thyromimetic activity. These results support the biologic relevance of the nuclear sites.     

A homozygous deletion in the c-erbA beta thyroid hormone receptor gene in a patient with generalized thyroid hormone resistance: isolation and characterization of the mutant receptor.

Different point mutations have been identified in the T3-binding domain of the c-erbA beta thyroid hormone receptor gene that are associated with variant phenotypes of generalized thyroid hormone resistance (GTHR). In most cases of GTHR, heterozygotes are affected; a single mutant allele results in the inhibition of the function of normal thyroid hormone receptors. We report here a novel genetic abnormality, a 3-basepair (bp) deletion in the T3-binding domain of the beta-receptor in a kindred, S, with GTHR. One patient, S1, was the product of a consanguineous union of two heterozygotes and was homozygous for this defect. Heterozygotes from kindred S harbored a CAC deletion at nucleotides 1295-1297, which resulted in the deduced loss of amino acid residue threonine at codon 332, and they displayed elevated free T4 levels and inappropriately normal TSH levels characteristic of other kindreds with GTHR. However, patient S1, who had two mutant alleles, had markedly elevated TSH and free T4 levels and displayed profound abnormalities in brain development and linear growth. A fibroblast c-erbA beta cDNA extending from codon 175 to stop codon 457 was cloned from patient S1, sequenced, and used to create a full-length mutant cDNA. The kindred S mutant receptor was synthesized in vitro and did not bind T3. This mutant receptor did bind with similar avidity as the wild-type human beta-receptor to thyroid hormone response elements of the human TSH beta (-12 to 43 bp) and rat GH (-188 to -160 bp) genes. Kindred S showed the effect in man of heterozygous and homozygous expression of a dominant negative form of c-erbA beta.     

Sequential up-regulation of thyroid hormone beta receptor, ornithine transcarbamylase, and carbamyl phosphate synthetase mRNAs in the liver of Rana catesbeiana tadpoles during spontaneous and thyroid hormone-induced metamorphosis.

During both spontaneous and thyroid hormone (TH)-induced metamorphosis, the Rana catesbeiana tadpole undergoes postembryonic developmental changes in its liver which are necessary for its transition from an ammonotelic larva to a ureotelic adult. Although this transition ultimately results from marked increases in the activities and/or de novo synthesis of the urea cycle enzymes, the precise molecular means by which TH exerts this tissue-specific response are presently unknown. Recent reports, using RNA from whole Xenopus laevis tadpole homogenates and indirect means of measuring TH receptor (TR) mRNAs, suggest a correlation between the up-regulation of TR beta-mRNAs and the general morphological changes occurring during amphibian metamorphosis. To assess whether or not this same relationship exists in a TH-responsive tissue, such as liver, we isolated and characterized a cDNA clone containing the complete nucleotide sequence for a R. catesbeiana urea cycle enzyme, ornithine transcarbamylase (OTC), as well as a genomic clone containing a portion of the hormone-binding domain of a R. catesbeiana TR beta gene. Through use of these homologous sequences and a heterologous cDNA fragment encoding rat carbamyl phosphate synthetase (CPS), we directly determined the relative levels of the TR beta, OTC, and CPS mRNAs in liver from spontaneous and TH-induced tadpoles. Our results establish that TH affects an up-regulation of mRNAs for its own receptor prior to up-regulating CPS and OTC mRNAs. Moreover, results with cultured tadpole liver demonstrate that TH, in the absence of any other hormonal influence, can affect an up-regulation of both the TR beta and OTC mRNAs.     

Influence of hormonal imprinting on hormone level. Permanent receptor damaging effect of pituitary hormones administered to newly-hatched cockerels

The overlapping effect of TSH and FSH on the gonad and on the thyroid gland can be demonstrated in cockerels even at the age of five weeks. These hormones influence the secretion of testosterone in a similar way and to a similar extent, while on the thyroxine level the influence of the specific hormone is more pronounced. Neonatal FSH and TSH treatment considerably decreased the basal testosterone level measured at the age of five weeks. Neonatal FSH treatment increased the basal T4 level while TSH treatment decreased it. The effect of TSH treatment administered at the age of five weeks in increasing the testosterone level was weakened after neonatal pretreatment with any iodine hormone. The effect of TSH treatment could only be inhibited by neonatal FSH pretreatment. Neonatal pretreatment with any of the trophormones caused a diminution of the T4 level augmenting of FSH and TSH administered at the age of five weeks.     

The effect of the juvenile hormone analog, fenoxycarb, on ecdysone receptor B1 expression in the midgut of Bombyx mori during larval-pupal metamorphosis

The Bombyx mori (Lepidoptera: Bombycidae) midgut undergoes remodeling during the larval-pupal metamorphosis. All metamorphic events in insects are controlled by mainly two hormones: 20-hydroxyecdysone (20E) and juvenile hormone (JH). Fenoxycarb, O-ethyl N-(2-(4-phenoxyphenoxy)-ethyl) carbamate, has been shown to be one of the most potent juvenile hormone analogs against a variety of insect species. In this study, the effect of fenoxycarb on EcR-B1 protein expression in the midgut of Bombyx mori during the remodeling processwas investigated. Fenoxycarb was topically treated to the beginning of the fifth instar Bombyx larvae. Its application prolonged the last instar and prevented metamorphic events. Analyses were performed from day 6 of the fifth instar to 24 hr after pupation in controls and to day 14 of the fifth instar in the fenoxycarb treated group. According to our results, the presence of EcR-B1 in the midguts of the fenoxycarb treated group during the feeding period suggested that EcR-B1 was involved in the functioning of larval cells and during this period fenoxycarb did not affect EcR-B1 status. Immediately after termination of the feeding stage, the amount of EcR-B1 protein increased, which indicated that it may strengthen the ecdysone signal for commitment of remodeling process. In the fenoxycarb treated group, its upregulation was delayed, which may be related to the inhibition of ecdysone secretion from the prothoracic gland.