In vitro and in silico approach to study the hormonal activities of the alternative plasticizer tri-(2-ethylhexyl) trimellitate TEHTM and its metabolites.

Tri-(2-ethylhexyl) trimellitate (TEHTM) is a plasticizer for polyvinyl chloride (PVC) material used in medical devices. It is an alternative to di-(2-ethylhexyl) phthalate (DEHP), a well-known reprotoxic and endocrine disruptor. As plasticizers are known to easily migrate when in contact with fatty biological fluids, patient exposure to TEHTM is highly probable. However, there is currently no data on the potential endocrine-disrupting effects of its human metabolites. To evaluate the effects of TEHTM metabolites on endocrine activity, they were first synthesized and their effects on estrogen, androgen and thyroid receptors, as well as steroid synthesis, were investigated by combining in vitro and in silico approaches. Among the primary metabolites, only 4-MEHTM (4-mono-(2-ethylhexyl) trimellitate) showed agonist activities on ERs and TRs, while three diesters were TR antagonists at non-cytotoxic concentrations. These results were completed by docking experiments which specified the ER and TR isoforms involved. A mixture of 2/1-MEHTM significantly increased the estradiol level and reduced the testosterone level in H295R cell culture supernatants. The oxidized secondary metabolites of TEHTM had no effect on ER, AR, TR receptors or on steroid hormone synthesis. Among the fourteen metabolites, these data showed that two of them (4-MEHTM and 2/1-MEHTM) induced effect on hormonal activities in vitro. However, by comparing the concentrations of the primary metabolites found in human urine with the active concentrations determined in bioassays, it can be suggested that the metabolites will not be active with regard to estrogen, androgen, thyroid receptors and steroidogenesis-mediated effects.

Pubertal fenvalerate exposure impairs cognitive and behavioral development partially through down-regulating hippocampal thyroid hormone receptor signaling.

Fenvalerate, a synthetic pyrethroid insecticide, is an environmental endocrine disruptor and neurodevelopmental toxicant. An early report found that pubertal exposure to high-dose fenvalerate impaired cognitive and behavioral development. Here, we aimed to further investigate the effect of pubertal exposure to low-dose fenvalerate on cognitive and behavioral development. Mice were orally administered with fenvalerate (0.2, 1.0 and 5.0 mg/kg) daily from postnatal day (PND) 28 to PND56. Learning and memory were assessed by Morris water maze. Anxiety-related activities were detected by open-field and elevated plus-maze. Increased anxiety activities were observed only in females exposed to fenvalerate. Spatial learning and memory were damaged only in females exposed to fenvalerate. Histopathology observed numerous scattered shrinking neurons and nuclear pyknosis in hippocampal CA1 region. Neuronal density was reduced in hippocampal CA1 region of fenvalerate-exposed mice. Mechanistically, hippocampal thyroid hormone receptor (TR)ß1 was down-regulated in a dose-dependent manner in females. In addition, TRα1 was declined only in females exposed to 5.0 mg/kg fenvalerate. Taken together, these suggests that pubertal exposure to low-dose fenvalerate impairs cognitive and behavioral development in a gender-dependent manner. Hippocampal TR signaling may be, at least partially, involved in fenvalerate-induced impairment of cognitive and behavioral development.

Di-(2-ethylhexyl) phthalate inhibits expression and internalization of transthyretin in human placental trophoblastic cells.

During pregnancy, fetal thyroid hormones (THs) are dependent on maternal placental transport and their physiological level is crucial for normal fetal neurodevelopment. Earlier research has shown that Di-(2-ethylhexyl) phthalate (DEHP) disrupts thyroid function and THs homeostasis in pregnant women and fetuses, and affects placental THs transport. However, the underlying mechanisms are poorly understood. The present study, therefore, aimed to systematically investigate the potential mechanisms of DEHP-induced disruption in the placental THs transport using two human placental trophoblastic cells, HTR-8/SVneo cells and JEG-3 cells. While the exposure of DEHP at the doses of 0-400 µM for 24 h did not affect cell viability, we found reduced consumption of T3 and T4 in the culture medium of HTR-8/Svneo cells treated with DEHP at 400 µM. DEHP treatment did not affect T3 uptake and the expression of monocarboxylate transporters 8 (MCT8) and organic anion transporters 1C1 (OATP1C1). However, DEHP significantly inhibited transthyretin (TTR) internalization, down-regulated TTR, deiodinase 2 (DIO2), and thyroid hormone receptors mRNA expression and protein levels, and up-regulated deiodinase 3 (DIO3) protein levels in a dose-dependent manner. These results indicate that DEHP acts on placental trophoblast cells, inhibits its TTR internalization, down-regulates TTR expression and affects the expression of DIO2, DIO3, and thyroid hormone receptor. These may be the mechanisms by which PAEs affects THs transport through placental.

Di(2-ethylhexyl)phthalate induces reproductive toxicity via JAZF1/TR4 pathway and oxidative stress in pubertal male rats.

Di(2-ethylhexyl)phthalate (DEHP) is a typical endocrine-disrupting chemical and reproductive toxicant. Although previous studies have attempted to describe the mechanism by which DEHP exposure results in reproductive dysfunction, few studies focused on puberty, a critical period of reproductive development, and the increased susceptibility to injury in adolescents. To elucidate the mechanism underpinning the testicular effects of DEHP in puberty, we sought to investigate the JAZF1/TR4 pathway in the testes of pubertal rats. Specifically, we focused on the role of the JAZF1/TR4 pathway in male reproduction, including the genes JAZF1, TR4, Sperm 1, and Cyclin A1. In the present study, rats were exposed to increasing concentrations of DEHP (0, 250, 500, and 1000 mg/kg/day) by oral gavages for 30 days. Then we assayed testicular zinc and oxidative stress levels. Our results indicated that DEHP exposure could lead to oxidative stress and decrease the contents of testicular zinc. Additionally, significant morphological changes and cell apoptosis were observed in testes exposed to DEHP, as identified by hematoxylin and eosin staining and the terminal deoxynucleotidyl transferase-mediated nick and labeling assay. By measuring the expression levels of the above relevant genes by qPCR, we found the DEHP-induced increased expression of JAZF1 and decreased expression of TR4, Sperm 1, and Cyclin A1. Therefore, we have demonstrated that in vivo exposure to DEHP might induce reproductive toxicity in pubertal male rats through the JAZF1/TR4 pathway and oxidative stress.

Evaluation of endocrine activities of ellagic acid and urolithins using reporter gene assays.

Urolithins are metabolites produced in the gut following consumption of ellagitannins and ellagic-acid-rich food, such as pomegranates, berries, and nuts. Compelling biological activities of urolithins together with variabilities between individuals in the metabolic capacity of the resident gut microbiota to produce urolithins, have suggested potential benefits of direct consumption of urolithins. Based on the structures of ellagic acid and urolithins, they might be expected to show endocrine effects. We report on their impact on the estrogen, androgen, glucocorticoid and thyroid-hormone receptors, as determined in vitro using reporter gene assays in the Hela9903 (estrogen receptor), MDA-kb2 (androgen and glucocorticoid receptors) and GH3.TRE-Luc (thyroid hormone receptor) cell lines. Urolithins A and B, but not ellagic acid and urolithin D, showed estrogenic activities on estrogen receptor subtype α under our assay conditions, with EC50 values of 5.59 µM and 32.60 µM, respectively. Moreover, ellagic acid and urolithins A and D showed anti-thyroid hormonal activities (IC50 values of 37.45 µM, 30.32 µM and 8.80 µM, respectively). Glucocorticoid and androgen agonist and antagonist activities were assessed using a luciferase reporter gene assay in the MDA-kb2 cell line. None of these tested compounds showed glucocorticoid agonist or antagonist activities, and ellagic acid showed weak androgen agonist activity, although only at the highest concentration tested. Detected estrogen and antithyroid activities warrant further risk assessment in relation to the exposure of urolithins in humans.

Cigarette Smoke Extract Disrupts Transcriptional Activities Mediated by Thyroid Hormones and Its Receptors.

Cigarette smoke contains over 4800 compounds, including at least 200 toxicants or endocrine disruptors. Currently, effects of cigarette smoke on thyroid hormone (TH) levels remains to be clarified. Here, we demonstrate that cigarette smoke extract (CSE) possesses thyroid hormone properties and acts synergistically as a partial agonist for thyroid hormone receptors (TRs) in the presence of TH. In transient gene expression experiments, CSE stimulated transcriptional activity with TH in a dose-dependent manner. Stimulatory effects were observed with physiological TH concentrations, although CSE did not activate TRs without TH. CSE (5%) dissolved in phosphate-buffered saline (PBS) supplemented with 1 nM TH was approximately comparable to 3.2±0.1 and 2.3±0.2 nM of TRα1 and TRß1, respectively. To illustrate probable mechanisms of the CSE agonistic activity, effects on TR mediated transcriptional functions with cofactors were investigated. With a mammalian two-hybrid assay, CSE recruited the nuclear coactivators glucocorticoid receptor interacting protein 1 (GRIP1) and steroid receptor coactivator 1 (SRC1) to the TR. Unsaturated carbonyl compounds, acrolein, crotonaldehyde, and methyl vinyl ketone, representative constituents of CSE, retained such agonistic properties and possibly contributed to stimulatory effects. The results suggest that CSE recruits a transcriptional activator and may reinforce TH binding to the TR additively, resulting in gene expression. CSE partially agonizes TH action and may disturb the function of various nuclear hormone receptor types and their cofactors to disrupt the physiological processes.

Assessment of endocrine disruption and oxidative potential of bisphenol-A, triclosan, nonylphenol, diethylhexyl phthalate, galaxolide, and carbamazepine, common contaminants of municipal biosolids.

The use of biosolids as a soil conditioner and fertiliser is hindered by the limited knowledge on the risks of micro-contaminants they contain. This study investigated the binding of six organic contaminants commonly found in biosolids, to the estrogen (ER), androgen (AR), aryl hydrocarbon (AhR), and transthyretin (TTR) receptors and their redox activity. Triclosan (TCS), bisphenol-A (BPA), and technical nonylphenol (TNP) had affinity for the TTR with relative potencies of 0.3, 0.03, and 0.076 respectively. Further, binding to TTR was the only toxicological response observed for carbamazepine, which induced sub-maximal response and relative potency of 0.0017. Estrogenic activity was induced by BPA, galaxolide (HHCB), diethylhexyl phthalate (DEHP) and TNP with BPA having the strongest potency of 5.1 × 10-6 relative to estradiol. Only BPA showed androgenic activity but it was not quantifiable. BPA also showed anti-androgenic activity along with TCS, HHCB, and TNP in the order of TNP > HHCB > TCS ~ BPA (relative potencies 0.126, 0.042, 0.032, 0.03). No compounds exhibited anti-estrogenic or AhR activity, or were redox-active in the dithiothreitol assay. The results highlight the multiple modes of action through which these compounds may impact exposed organisms, and the concentrations at which effects may occur. This allows assessment of the likelihood of effects being observed at environmental concentrations, and the potential contribution of these compounds.

Identification and Molecular Interaction Studies of Thyroid Hormone Receptor Disruptors among Household Dust Contaminants.

Thyroid hormone disrupting chemicals (THDCs), often found abundantly in the environment, interfere with normal thyroid hormone signaling and induce physiological malfunctions, possibly by affecting thyroid hormone receptors (THRs). Indoor dust ingestion is a significant human exposure route of THDCs, raising serious concerns for human health. Here, we developed a virtual screening protocol based on an ensemble of X-ray crystallographic structures of human THRß1 and the generalized Born solvation model to identify potential THDCs targeting the human THRß1 isoform. The protocol was applied to virtually screen an in-house indoor dust contaminant inventory, yielding 31 dust contaminants as potential THRß1 binders. Five predicted binders and one negative control were tested using isothermal titration calorimetry, of which four, i.e., 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), bisphenol A (3-chloro-2-hydroxypropyl) (2,3-dihydroxypropyl) ether (BADGE-HCl-H2O), 2,2',4,4'-tetrahydroxybenzophenone (BP2), and 2,4-dichlorophenoxyacetic acid (2,4-D), were identified as THRß1 binders with binding affinities ranging between 60 µM and 460 µM. Molecular dynamics (MD) simulations were employed to examine potential binding modes of these binders and provided a rationale for explaining their specific recognition by THRß1. The combination of in vitro binding affinity measurements and MD simulations allowed identification of four new potential THR-targeting THDCs that have been found in household dust. We suggest using the developed structure-based virtual screening protocol to identify and prioritize testing of potential THDCs.

Endocrine-Disrupting Activity of Hydraulic Fracturing Chemicals and Adverse Health Outcomes After Prenatal Exposure in Male Mice.

Oil and natural gas operations have been shown to contaminate surface and ground water with endocrine-disrupting chemicals. In the current study, we fill several gaps in our understanding of the potential environmental impacts related to this process. We measured the endocrine-disrupting activities of 24 chemicals used and/or produced by oil and gas operations for five nuclear receptors using a reporter gene assay in human endometrial cancer cells. We also quantified the concentration of 16 of these chemicals in oil and gas wastewater samples. Finally, we assessed reproductive and developmental outcomes in male C57BL/6J mice after the prenatal exposure to a mixture of these chemicals. We found that 23 commonly used oil and natural gas operation chemicals can activate or inhibit the estrogen, androgen, glucocorticoid, progesterone, and/or thyroid receptors, and mixtures of these chemicals can behave synergistically, additively, or antagonistically in vitro. Prenatal exposure to a mixture of 23 oil and gas operation chemicals at 3, 30, and 300 µg/kg · d caused decreased sperm counts and increased testes, body, heart, and thymus weights and increased serum testosterone in male mice, suggesting multiple organ system impacts. Our results suggest possible adverse developmental and reproductive health outcomes in humans and animals exposed to potential environmentally relevant levels of oil and gas operation chemicals.

Chlorotriazines do not activate the aryl hydrocarbon receptor, the oestrogen receptor or the thyroid receptor in in vitro assays.

Atrazine, prometryn, propazine and simazine are chlorotriazines that are commonly employed as herbicides. However, their use is a major cause of concern, due to their reported endocrine disrupting effects in different taxa. Data from studies on the molecular and cellular processes underlying the hormonal action of these substances are contradictory. The ability of these chlorotriazines and the atrazine metabolites, desethyl-s-chlorotriazine and desisopropyl-s-chlorotriazine, to trigger responses mediated by the oestrogen receptor (ER), aryl hydrocarbon receptor (AhR) and thyroid receptor (TR), was studied by using in vitro approaches. Transcriptional activation assays were applied to observe the activation of ER and TR. The induction of ethoxyresorufin-O-deethylase (EROD) activity in the RTG-2 cell line served as an indicator of AhR activation. No responses were found in any of the assays, with any of the six chlorotriazines tested. Our observations indicate that the chlorotriazines tested are unlikely to cause their endocrine effects via these receptors.

6-Mercaptopurine augments glucose transport activity in skeletal muscle cells in part via a mechanism dependent upon orphan nuclear receptor NR4A3.

The purine anti-metabolite 6-mercaptopurine (6-MP) is widely used for the treatment of leukemia and inflammatory diseases. The cellular effects of 6-MP on metabolism remain unknown; however, 6-MP was recently found to activate the orphan nuclear receptor NR4A3 in skeletal muscle cell lines. We have reported previously that NR4A3 (also known as NOR-1, MINOR) is a positive regulator of insulin sensitivity in adipocytes. To further explore the role of NR4A3 activation in insulin action, we explored whether 6-MP activation of NR4A3 could modulate glucose transport system activity in L6 skeletal muscle cells. We found that 6-MP increased both NR4A3 expression and NR4A3 transcriptional activity and enhanced glucose transport activity via increasing GLUT4 translocation in both basal and insulin-stimulated L6 cells in an NR4A3-dependent manner. Furthermore, 6-MP increased levels of phospho-AS160, although this effect was not modulated by NR4A3 overexpression or knockdown. These primary findings provide a novel proof of principle that 6-MP, a small molecule NR4A3 agonist, can augment glucose uptake in insulin target cells, although this occurs via both NR4A3-dependent and -independent actions; the latter is related to an increase in phospho-AS160. These results establish a novel target for development of new treatments for insulin resistance.

[Thyroid hormones and their precursors I. Biochemical properties]. / Pajzsmirigyhormonok és eloanyagaik I. Biokémiai tulajdonságok.

This paper and the following one (see the next issue of Acta Pharmaceutica Hungarica) survey the biological roles and the related site-specific physico-chemical parameters (basicity and lipophilicity) of the presently known thyroid hormones (thyroxine, liothyronine and reverse liothyronine) and their biological precursors (monoiodotyrosine and diiodotyrosine). Here the literature of the thyroid hormone biochemistry, biosynthesis, plasma- and membrane transport is summarized, focusing on the pH-dependent processes. Biosyntheses of the thyroid hormones take place by oxidative coupling of two iodotyrosine residues catalyzed by thyreoperoxidase in thyreoglobulin. The protonation state of the precursors, especially that of the phenolic OH is crucial for the biosynthesis, since anionic iodotyrosine residues can only be coupled in the thyroid hormone biosyntheses. In the blood more than 99% of the circulating thyroid hormone is bound to plasma proteins among which the thyroxine-binding globulin and transthyretin are crucial. The amphiphilic character of the hormones is assumed to be the reason why their membrane transport is an energy-dependent, transport-mediated process, in which the organic anion transporter family, mainly OATP1C1, and the amino acid transporters, such as MCT8 play important roles. Liothyronine is the biologically active hormone; it binds the thyroid hormone receptor, a type of nuclear receptor. There are two major thyroid hormone receptor (TR) isoforms, alfa (TRalpha) and beta (TRbeta). The activation of the TRalpha is associated with modifications in cardiac behavior, while activation of the TRbeta is associated with increasing metabolic rates, resulting in weight loss and reduction of blood plasma lipid levels. The affinity of the thyroid hormones for different proteins depends on the ionization state of the ligands. The site-specific physico-chemical characterization of the thyroid hormones is of fundamental importance to understand their (patho)physiological behavior and also, to influence their therapeutic properties at the molecular level.

Screening for potential effects of endocrine-disrupting chemicals in peri-urban creeks and rivers in Melbourne, Australia using mosquitofish and recombinant receptor-reporter gene assays.

Sexually mature male mosquitofish (Gambusia holbrooki) were collected from various sites around Melbourne in 2009 to evaluate the performance of gonopodial indices as a biomarker for endocrine disruption in Melbourne's waterways. The mosquitofish indices assessed were body length (BL), gonopodial length (GL)/BL ratio, ray 4:6 ratio and the absence or presence of hooks and serrae, and these varied between sites. The study was complemented by measurements of estrogenic, retinoid, thyroid and aryl hydrocarbon (AhR) receptor activities of the water. Male mosquitofish were 16.3-21.5 mm in length, and although there was a statistically significant positive relationship showing that bigger fish had longer gonopodia than small fish (r2 = 0.52, p < 0.001), there were few significant differences in GL/BL ratio of fish between sites. Measured estrogenic activity was mostly in the range 0.1-1.7 ng/L EEQ, with one site having much higher levels (~12 ng/L EEQ). Aryl hydrocarbon (AhR) receptor activity was observed in all water samples (7-180 ng/L ßNF EQ), although there was no consistent pattern in the level of AhR activity observed, i.e., 'clean' sites were as likely to return a high AhR activity response as urban or wastewater treatment plant (WWTP)-impacted sites. There was no correlation between measurements of receptor actvity and gonopodial length (GL):BL ratio and BL. We conclude that the mosquitofish gonopodia only fulfills part of the criteria for biomarker selection for screening. The mosquitofish indices assessed were cheap and easy-to-perform procedures; however, there is no baseline data from the selected sites to evaluate whether differences in the morpholical indices observed at a site were a result of natural selection in the population or due to estrogenic exposure.

Thyroid disruption in the lizard Podarcis bocagei exposed to a mixture of herbicides: a field study.

Pesticide exposure has been related with thyroid disrupting effects in different vertebrate species. However, very little is known about the effects of these compounds in reptiles. In the Mediterranean area, lacertid lizards are the most abundant vertebrate group in agroecosystems, and have been identified as potential model species for reptile ecotoxicology. The aim of this study was to understand if the herbicides applied in corn fields have thyroid disruptive effects in the lizard Podarcis bocagei. Adult male lizards were captured in north-western Portugal in corn fields treated with herbicides (exposed sites), and in organic agricultural fields (reference sites). Thyroid and male gonad morphology and functionality, and testosterone levels were investigated through histological, immunohistochemical and biochemical techniques. Lizards from exposed locations displayed thyroid follicular lumens with more reabsorption vacuoles and significantly larger follicular area than those from reference fields. Furthermore, testes of lizards from exposed locations had significantly larger seminiferous tubule diameters, significantly higher number of spermatogenic layers and displayed an up-regulation of thyroid hormone receptors when compared with lizards from reference areas. These findings strongly suggest that the complex mixture of herbicides that lizards are exposed to in agricultural areas have thyroid disrupting effects which ultimately affect the male reproductive system. Alachlor, which has demonstrated thyroid effects in mammals, may be largely responsible for the observed effects.

Receptors for thyrotropin-releasing hormone, thyroid-stimulating hormone, and thyroid hormones in the macaque uterus: effects of long-term sex hormone treatment.

OBJECTIVE: Thyroid gland dysfunction is associated with menstrual cycle disturbances, infertility, and increased risk of miscarriage, but the mechanisms are poorly understood. However, little is known about the regulation of these receptors in the uterus. The aim of this study was to determine the effects of long-term treatment with steroid hormones on the expression, distribution, and regulation of the receptors for thyrotropin-releasing hormone (TRHR) and thyroid-stimulating hormone (TSHR), thyroid hormone receptor α1/α2 (THRα1/α2), and THRß1 in the uterus of surgically menopausal monkeys. METHODS: Eighty-eight cynomolgus macaques were ovariectomized and treated orally with conjugated equine estrogens (CEE; n = 20), a combination of CEE and medroxyprogesterone acetate (MPA; n = 20), or tibolone (n = 28) for 2 years. The control group (OvxC; n = 20) received no treatment. Immunohistochemistry was used to evaluate the protein expression and distribution of the receptors in luminal epithelium, glands, stroma, and myometrium of the uterus. RESULTS: Immunostaining of TRHR, TSHR, and THRs was detected in all uterine compartments. Epithelial immunostaining of TRHR was down-regulated in the CEE + MPA group, whereas in stroma, both TRHR and TSHR were increased by CEE + MPA treatment as compared with OvxC. TRHR immunoreactivity was up-regulated, but THRα and THRß were down-regulated, in the myometrium of the CEE and CEE + MPA groups. The thyroid-stimulating hormone level was higher in the CEE and tibolone groups as compared with OvxC, but the level of free thyroxin did not differ between groups. CONCLUSIONS: All receptors involved in thyroid hormone function are expressed in monkey uterus, and they are all regulated by long-term steroid hormone treatment. These findings suggest that there is a possibility of direct actions of thyroid hormones, thyroid-stimulating hormone and thyrotropin-releasing hormone on uterine function.

Regulation of thyroid hormone sensitivity by differential expression of the thyroid hormone receptor during Xenopus metamorphosis.

During amphibian metamorphosis, a series of dynamic changes occur in a predetermined order. Hind limb morphogenesis begins in response to low levels of thyroid hormone (TH) in early prometamorphosis, but tail muscle cell death is delayed until climax, when TH levels are high. It takes about 20 days for tadpoles to grow from early prometamorphosis to climax. To study the molecular basis of the timing of tissue-specific transformations, we introduced thyroid hormone receptor (TR) expression constructs into tail muscle cells of Xenopus tadpoles. The TR-transfected tail muscle cells died upon exposure to a low level of thyroxine (T4). This cell death was suggested to be mediated by type 2 iodothyronine deiodinase (D2) that converts T4 to T3-the more active form of TH. D2 mRNA was induced in the TR-overexpressing cells by low levels of TH. D2 promoter contains a TH-response element (TRE) with a lower affinity for TR. These results show that the TR transfection confers the ability to respond to physiological concentrations of TH at early prometamorphosis to tail muscle cells through D2 activity and promotes TH signaling. We propose the positive feedback loop model to amplify the cell's ability to respond to low levels of T4.

Cocaine decreases expression of neurogranin via alterations in thyroid receptor/retinoid X receptor signaling.

Mounting evidence suggests a potential link between cocaine abuse, disruptions in hypothalamic-pituitary-thyroid axis signaling, and neuroplasticity, but molecular mechanisms remain unknown. Neurogranin (Ng) is a gene containing a thyroid hormone-responsive element within its first intron that is involved in synaptic plasticity. Transcriptional activation requires heterodimerization of thyroid hormone receptor (TR) and retinoid X receptor (RXR) bound by their respective ligands, tri-iodothryonine and 9-cis-retinoic acid (9-cis-RA), and subsequent binding of this complex to the thyroid hormone-responsive element of the Ng gene. In this study, the effects of chronic cocaine abuse on Ng expression in euthyroid and hypothyroid mice were assessed. In cocaine-treated mice, decreased Ng expression was observed in the absence of changes in levels of thyroid hormones or other hypothalamic-pituitary-thyroid signaling factors. Therefore, we hypothesized that cocaine decreases Ng expression via alterations in 9-cis-RA availability and TR/RXR signaling. In support of this hypothesis, RXR-γ was significantly decreased in brains of cocaine-treated mice while CYP26A1, the main enzyme responsible for neuronal RA degradation, was significantly increased. Results from this study provide the first evidence for a direct effect of cocaine abuse on TR/RXR signaling, RA metabolism, and transcriptional regulation of Ng, a gene essential for adult neuroplasticity.

Identical gene regulation patterns of T3 and selective thyroid hormone receptor modulator GC-1.

Synthetic selective thyroid hormone (TH) receptor (TR) modulators (STRM) exhibit beneficial effects on dyslipidemias in animals and humans and reduce obesity, fatty liver, and insulin resistance in preclinical animal models. STRM differ from native TH in preferential binding to the TRß subtype vs. TRα, increased uptake into liver, and reduced uptake into other tissues. However, selective modulators of other nuclear receptors exhibit important gene-selective actions, which are attributed to differential effects on receptor conformation and dynamics and can have profound influences in animals and humans. Although there are suggestions that STRM may exhibit such gene-specific actions, the extent to which they are actually observed in vivo has not been explored. Here, we show that saturating concentrations of the main active form of TH, T(3), and the prototype STRM GC-1 induce identical gene sets in livers of euthyroid and hypothyroid mice and a human cultured hepatoma cell line that only expresses TRß, HepG2. We find one case in which GC-1 exhibits a modest gene-specific reduction in potency vs. T(3), at angiopoietin-like factor 4 in HepG2. Investigation of the latter effect confirms that GC-1 acts through TRß to directly induce this gene but this gene-selective activity is not related to unusual T(3)-response element sequence, unlike previously documented promoter-selective STRM actions. Our data suggest that T(3) and GC-1 exhibit almost identical gene regulation properties and that gene-selective actions of GC-1 and similar STRM will be subtle and rare.

Effect of 2,2',4,4'-tetrahydroxybenzophenone (BP2) on steroidogenesis in testicular Leydig cells.

Endocrine disruptors (EDs) affect the function of animal reproductive systems. Recently, 2,2',4,4'-tetrahydroxybenzophenone (BP2), which is a component of UV protection products, was found to be an ED that interferes with the thyroid hormone (TH) axis. However, BP2 activity in the testis has not been well addressed. In this study, we have examined the effects of BP2 on steroidogenesis in testicular Leydig cells in connection with thyroid hormone signaling, which is known to play an important role in testicular development and function. Our study showed that BP2 affected the expression of steroidogenic enzyme genes in testicular Leydig cells, which is differentially regulated by thyroid hormone/thyroid hormone receptor (TR) signaling. In MA-10 Leydig cell line, TR/T3 signaling increased the expression of P450c17 and P450scc, while it decreased the expression of StAR and 3ß-HSD. Interestingly, BP2 affected the expression of steroidogenic enzyme genes in a manner opposite to that of T3 signaling. BP2 downregulated the TRα/T3-activation of P450c17 and P450scc expression while enhancing the TRα/T3-repression of StAR and 3ß-HSD expression. Transient transfection analyses with promoter-reporter constructs revealed that BP2 altered the expression of steroidogenic enzyme genes by affecting the cAMP and Nur77-activated promoter activity of P450c17, StAR, and 3ß-HSD. Animal experiments with mice revealed that BP2 decreased the production of testosterone in the testis by affecting the expression of some steroidogenic enzyme genes in vivo. Together, these findings elucidate a molecular mechanism of BP2 action underlying testicular steroidogenesis and also suggest that BP2 acts, in part, as a thyroid antagonist that affects steroidogenesis in the testis.

Thyroid hormone receptor-mediated transcription is suppressed by low dose phthalate.

SUMMARY: Phthalates are synthetic chemicals used mainly as solvents, additives and plasticizers in polyvinylchloride (PVC) products to increase their flexibility. Phthalate plasticizers are not chemically bound to PVC, so they easily leach into the environment. There is currently heightened concern about potential health risk, especially endocrine disrupting effects associated with the use of these chemicals. We therefore investigated the effects of phthalate on thyroid hormone receptor (TR)-mediated transcription using transient transfection studies and found that low dose phthalate (10-7) M suppressed thyroid hormone (TH)-induced TR-mediated transcription by 30%. We further examined the effect of phthalate on TR-thyroid hormone response element (TRE) binding, and found no dissociation of TR from TRE. Phthalate did not also dissociate coactivator (steroid receptor coactivator-1) from TR neither did it recruit corepressor (nuclear corepressor; NCoR) to TR in the presence of TH. Our results indicate that low phthalate can disrupt TR-mediated gene expression and interfere with TH balance in TH-sensitive organs including the developing brain.