T4-mediated rescue of aortic malformations in hypothyroid rats indicates maternal thyroid status can affect great vessel development.

Pexacerfont is a corticotrophin-releasing factor subtype 1 receptor (CRF-1) antagonist developed for potential treatment of anxiety and stress-related disorders. In male rats, pexacerfont caused hepatic enzyme induction leading to increased thyroxine (T4) clearance. When administered to pregnant rats on gestation day 6 to 15, pexacerfont at 300 mg/kg/day (30× mean AUC in humans at 100 mg/day) produced similar effects on thyroid homeostasis with serum T4 and thyroid-stimulating hormone levels that were 0.3-0.5× and 3.3-3.7× of controls, respectively. At this dose, fetuses of pexacerfont-treated dams presented findings associated with maternal hypothyroidism including growth retardation and increased skeletal alterations. Additionally, there were unexpected great vessel malformations that were mostly derived from the 4th pharyngeal arch artery in 5 (4.3%) fetuses from 3 (15.8%) litters. The etiology was unclear whether the vascular malformations were related to insufficient thyroid hormones or another mechanism. To better understand this relationship, pregnant rats were implanted with a subcutaneous L-thyroxine pellet designed to provide a sustained release of T4 throughout organogenesis in rat embryos (GD 6 to 15; the dosing period of pexacerfont). T4 supplementation produced a near euthyroid state in pexacerfont-treated dams and completely prevented the fetal vascular malformations. These results suggest maternal T4 levels during organogenesis may have a role in great vessel morphogenesis associated with patterning and/or regression of pharyngeal arch arteries. Although previous clinical reports have speculated a potential relationship between thyroid hormone homeostasis and early cardiovascular development, this is the first report to experimentally demonstrate this relationship in great vessel morphogenesis.

Glucocorticoid and progesterone mechanisms in photoreceptor survival.

Death of retinal photoreceptors is the basis of prevalent blinding diseases. Since steroids might have a therapeutic role in retinal degenerations, we compared the protective effects of dexamethasone and progesterone on photoreceptor death induced by mifepristone and light exposure. Therefore, we studied the effective protection doses for each steroid in the two models. In addition, we analyzed changes in the levels of pro- and antiapoptotic molecules, glucocorticoid receptors α and ß (GRα and GRß), and rhodopsin under conditions of successful protection and photoreceptor survival. Mifepristone and light exposure selectively damaged photoreceptors. In light exposed retinas, photoreceptors mainly disappeared in the dorsotemporal region, while mifepristone produced a uniform damage. Dexamethasone and progesterone, at the same dose of 4 mg/kg/day for 2 days, preserved over 88% photoreceptor nuclei in both models. Assessment of cell death regulators showed that, in control retinas, both steroids activated BCL-XL, a prosurvival molecule, and decreased BID, a proapoptotic regulator. After steroid treatment of damaged retinas, BCL-XL, BCL2 and BAX showed characteristic patterns depending on the use of dexamethasone or progesterone on mifepristone or light exposed retinas. By contrast, BID decreased with any injury-steroid combination. Changes in GRα or GRß levels did not correlate with survival but were consistent with a mechanism of ligand induced downregulation of receptor expression. GRß might be upregulated by progesterone. Both dexamethasone and progesterone increased retinal rhodopsin stores, suggesting a link between photoreceptor protection and transduction pathways. Results show that dexamethasone and progesterone induced comparable but not identical protection responses in each model.

The melatonin receptor antagonist luzindole induces the activation of cellular stress responses and decreases viability of rat pancreatic stellate cells.

In this study, we have examined the effects of luzindole, a melatonin receptor-antagonist, on cultured pancreatic stellate cells. Intracellular free-Ca2+ concentration, production of reactive oxygen species (ROS), activation of mitogen-activated protein kinases (MAPK), endoplasmic reticulum stress and cell viability were analyzed. Stimulation of cells with the luzindole (1, 5, 10 and 50 µm) evoked a slow and progressive increase in intracellular free Ca2+ ([Ca2+ ]i ) towards a plateau. The effect of the compound on Ca2+ mobilization depended on the concentration used. Incubation of cells with the sarcoendoplasmic reticulum Ca2+ -ATPase inhibitor thapsigargin (1 µm), in the absence of Ca2+ in the extracellular medium, induced a transient increase in [Ca2+ ]i . In the presence of thapsigargin, the addition of luzindole to the cells failed to induce further mobilization of Ca2+ . Luzindole induced a concentration-dependent increase in ROS generation, both in the cytosol and in the mitochondria. This effect was smaller in the absence of extracellular Ca2+ . In the presence of luzindole the phosphorylation of p44/42 and p38 MAPKs was increased, whereas no changes in the phosphorylation of JNK could be noted. Moreover, the detection of the endoplasmic reticulum stress-sensor BiP was increased in the presence of luzindole. Finally, viability was decreased in cells treated with luzindole. Because cellular membrane receptors for melatonin have not been detected in pancreatic stellate cells, we conclude that luzindole could exert direct effects that are not mediated through its action on melatonin membrane receptors.

Amyloid-ß induces sleep fragmentation that is rescued by fatty acid binding proteins in Drosophila.

Disruption of sleep/wake activity in Alzheimer's disease (AD) patients significantly affects their quality of life and that of their caretakers and is a major contributing factor for institutionalization. Levels of amyloid-ß (Aß) have been shown to be regulated by neuronal activity and to correlate with the sleep/wake cycle. Whether consolidated sleep can be disrupted by Aß alone is not well understood. We hypothesize that Aß42 can increase wakefulness and disrupt consolidated sleep. Here we report that flies expressing the human Aß42 transgene in neurons have significantly reduced consolidated sleep compared with control flies. Fatty acid binding proteins (Fabp) are small hydrophobic ligand carriers that have been clinically implicated in AD. Aß42 flies that carry a transgene of either the Drosophila Fabp or the mammalian brain-type Fabp show a significant increase in nighttime sleep and long consolidated sleep bouts, rescuing the Aß42-induced sleep disruption. These studies suggest that alterations in Fabp levels and/or activity may be associated with sleep disturbances in AD. Future work to determine the molecular mechanisms that contribute to Fabp-mediated rescue of Aß42-induced sleep loss will be important for the development of therapeutics in the treatment of AD. © 2016 Wiley Periodicals, Inc.

GLP-2 as Beneficial Factor in the Glucose Homeostasis in Mice Fed a High Fat Diet.

Glucagon like peptide-2 (GLP-2) is a gastrointestinal hormone released in response to dietary nutrients, which acts through a specific receptor, the GLP-2 receptor (GLP-2R). The physiological effects of GLP-2 are multiple, involving also the intestinal adaptation to high fat diet (HFD). In consideration of the well-known relationship between chronic HFD and impaired glucose metabolism, in the present study we examined if the blocking of the GLP-2 signaling by chronic treatment with the GLP-2R antagonist, GLP-2 (3-33), leads to functional consequences in the regulation of glucose metabolism in HFD-fed mice. Compared with animals fed standard diet (STD), mice at the 10th week of HFD showed hyperglycaemia, glucose intolerance, high plasma insulin level after glucose load, increased pancreas weight and ß cell expansion, but not insulin resistance. In HFD fed mice, GLP-2 (3-33) treatment for 4 weeks (from the 6th to the 10th week of diet) did not affect fasting glycaemia, but it significantly increased the glucose intolerance, both fasting and glucose-induced insulin levels, and reduced the sensitivity to insulin leading to insulin-resistance. In GLP-2 (3-33)-treated HFD mice pancreas was significantly heavier and displayed a significant increase in ß-cell mass in comparison with vehicle-treated HFD mice. In STD mice, the GLP-2 (3-33) treatment did not affect fasted or glucose-stimulated glycemia, insulin, insulin sensitivity, pancreas weight and beta cell mass. The present study suggests that endogenous GLP-2 may act as a protective factor against the dysregulation of the glucose metabolism that occurs in HFD mice, because GLP-2 (3-33) worsens glucose metabolism disorders.

Glucocorticoid receptor antagonism disrupts the reconsolidation of social reward-related memories in rats.

Reconsolidation is the process whereby consolidated memories are destabilized upon retrieval and restabilized to persist for later use. Although the neurobiology of the reconsolidation of both appetitive and aversive memories has been intensively investigated, reconsolidation of memories of physiologically relevant social rewards has received little attention. Social play, the most characteristic social behaviour displayed by young mammals, is highly rewarding, illustrated by the fact that it can induce conditioned place preference (CPP). Here, we investigated the role of signalling mechanisms implicated in memory processes, including reconsolidation, namely glucocorticoid, mineralocorticoid, NMDA glutamatergic and CB1 cannabinoid receptors, in the reconsolidation of social play-induced CPP in rats. Systemic treatment with the glucocorticoid receptor antagonist mifepristone before, but not immediately after, retrieval disrupted the reconsolidation of social play-induced CPP. Mifepristone did not affect social play-induced CPP in the absence of memory retrieval. Treatment with the NMDA receptor antagonist MK-801 modestly affected the reconsolidation of social play-induced CPP. However, the reconsolidation of social play-induced CPP was not affected by treatment with the mineralocorticoid and CB1 cannabinoid receptor antagonists spironolactone and rimonabant, respectively. We conclude that glucocorticoid neurotransmission mediates the reconsolidation of social reward-related memories in rats. These data indicate that the neural mechanisms of the reconsolidation of social reward-related memories only partially overlap with those underlying the reconsolidation of other reward-related memories.

[Glyphosate and its formulations--toxicity, occupational and environmental exposure]. / Glifosat i jego preparaty--toksycznosc, narazenie zawodowe i srodowiskowe.

Glyphosate (N-(phosphonomethyl)glycine) is an active ingredient of the most widely used herbicide formulations in protecting agricultural and horticultural crops. Numerous results (mostly published in the years 2010-2013) concerning the action of glyphosate and its formulations in the recent decade were analyzed. Initial reports about alleged biodegradability of glyphosate in the environment turned out to be wrong. It has been shown that glyphosate remains in the soil and can reach people by spreading along with groundwater. Recent publications have shown that glyphosate is detected at low concentrations in the human blood. Publications cited in this article, which indicate a possible induction of neoplastic changes by glyphosate formulation, have raised great concern and controversy in the scientific world. Presenting adverse effects of glyphosate and its formulations we focused on the role of glyphosate formulations in hormonal disorders by impeding the expression of steroidogenic acute regulatory protein and the inhibition of aromatase activity. The impact of glyphosate on oxygen reactive species formation, changes in redox system and the effect on necrosis and apoptosis in various types of cells was shown. We also revealed that glyphosate as a phosphonate herbicide does not inhibit directly the activity of acetylcholinesterase. Based on numerous studies it was noted that commercial formulations of glyphosate exhibit higher toxicity than that of the active substance itself. The discussed problems clearly show the need to evaluate the toxicity of glyphosate and its formulations and related potential threat to humans.

Potential hazards to embryo implantation: A human endometrial in vitro model to identify unwanted antigestagenic actions of chemicals.

Embryo implantation is a crucial step in human reproduction and depends on the timely development of a receptive endometrium. The human endometrium is unique among adult tissues due to its dynamic alterations during each menstrual cycle. It hosts the implantation process which is governed by progesterone, whereas 17ß-estradiol regulates the preceding proliferation of the endometrium. The receptors for both steroids are targets for drugs and endocrine disrupting chemicals. Chemicals with unwanted antigestagenic actions are potentially hazardous to embryo implantation since many pharmaceutical antiprogestins adversely affect endometrial receptivity. This risk can be addressed by human tissue-specific in vitro assays. As working basis we compiled data on chemicals interacting with the PR. In our experimental work, we developed a flexible in vitro model based on human endometrial Ishikawa cells. Effects of antiprogestin compounds on pre-selected target genes were characterized by sigmoidal concentration-response curves obtained by RT-qPCR. The estrogen sulfotransferase (SULT1E1) was identified as the most responsive target gene by microarray analysis. The agonistic effect of progesterone on SULT1E1 mRNA was concentration-dependently antagonized by RU486 (mifepristone) and ZK137316 and, with lower potency, by 4-nonylphenol, bisphenol A and apigenin. The negative control methyl acetoacetate showed no effect. The effects of progesterone and RU486 were confirmed on the protein level by Western blotting. We demonstrated proof of principle that our Ishikawa model is suitable to study quantitatively effects of antiprogestin-like chemicals on endometrial target genes in comparison to pharmaceutical reference compounds. This test is useful for hazard identification and may contribute to reduce animal studies.

The effects of prolactin receptor blockade in a murine endometriosis interna model.

Endometriosis in an estrogen-dependent disease that is characterized by the presence of endometrial tissue outside the uterine cavity leading to pain and infertility in many affected women. Highly efficient treatment options which create a hypo-estrogenic environment can cause side effects such as hot flushes and bone mass loss that are not favorable for premenopausal women. Previous work has demonstrated that increased local or systemic prolactin seems to be involved in the pathogenesis of endometriosis. Here we examined two prolactin receptor (PRLR) blocking antibodies in a murine endometriosis interna model which relies on the induction of systemic hyperprolactinemia in female SHN mice. The severity of the disease is determined by the degree of endometrial invasion into the myometrium. In this model, endometriosis was inhibited by clinical gold standards such as progestins and anti-estrogenic approaches. PRLR blockade completely inhibited endometriosis in this mouse model to the same extent as the anti-estrogen faslodex or the GnRH antagonist cetrorelix. In contrast to cetrorelix and faslodex, the PRLR antibodies did not decrease relative uterine weights and were thus devoid of anti-estrogenic effects. We therefore hypothesize that PRLR antibodies may present a novel and highly efficient treatment option for endometriosis with a good safety and tolerability profile. Clinical studies are on the way to test this hypothesis.

Finding maximal transcriptome differences between reprotoxic and non-reprotoxic phthalate responses in rat testis.

The chemical legislation of the EU, Registration, Evaluation, and Authorization of Chemicals (REACH), stipulates that about 30 000 chemical substances are to be assessed on their possible risks. Toxicological evaluation of these compounds will at least partly be based on animal testing. In particular, the assessment of reproductive toxicity is a very complicated, time-consuming and animal-demanding process. Introducing microarray-based technologies can potentially refine in vivo toxicity testing. If compounds of a distinct chemical class induce reproducible gene-expression responses with a recognizable overlap, these gene-expression signatures may indicate intrinsic features of certain compounds, including specific toxicity. In the present study, we have set out the first steps towards this approach for the reproductive toxicity of phthalates. Male rats were treated with a single dose of either reprotoxic or non-reprotoxic phthalates, and were analyzed 24 h afterwards. Subsequently, histopathological and gene-expression profiling analyses were performed. Despite ambiguous histopathological observations, we were able to identify genes with differential expression profiles between the reprotoxic phthalates and the non-reprotoxic counterparts. This shows that differences in gene-expression profiles, indicative of the type of exposure, may be detected earlier, or at lower doses, than classical pathological endpoints. These findings are promising for 'early warning' biomarker analyses and for using toxicogenomics in a category approach. Ultimately, this could lead to a more cost-effective approach for prioritizing the toxicity testing of large numbers of chemicals in a short period of time in hazard assessment of chemicals, which is one of the objectives of the REACH chemical legislation.

New in vitro reporter gene bioassays for screening of hormonal active compounds in the environment.

Identification of chemicals with endocrine-disrupting activities in the past two decades has led to the need for sensitive assays for detection and monitoring of these activities in the environment. In vitro reporter gene assays represent a relatively fast and easy-to-perform method for detection of compounds that are able to bind to hormonal receptors and stimulate or silence their transactivation activity, thus interfering with the hormone signaling pathways. This paper reviews upgrades on reporter gene assays performed during the last decade. The utilization of new reporter genes (luciferase and green fluorescent protein coding genes) significantly improved the sensitivity of the tests and made them faster. Reporter gene assays now represent a high-throughput system for screening chemicals for hormonal activity. Finally, modification of test set-ups for testing anti-hormonal activities also enabled measurements of endocrine-disrupting activities in complex environmental samples such as sediments and wastewater treatment plant effluents.

The effects of selected phenol and phthalate derivatives on steroid hormone production by cultured porcine granulosa cells.

We have investigated the effects of several phenols (octylphenol [OP], nonylphenol [NP], tert-octylphenol [tOP]) and phthalates (dioctylphthalate [DOP], diisodecylphthalate [DiDP], diisononylphthalate [DiNP]) on steroid hormone production by porcine ovarian granulosa cells after a 72-hour incubation. These chemicals are widely used as plasticisers and are suspected to possess endocrine disrupting properties. No changes were exhibited in basal progesterone production after treatment with NP or tOP, or with the tested phthalates. However, OP tended to decrease progesterone levels, while DOP and DiDP, at the lowest concentration used (10(-8)M), increased progesterone levels in the culture media. Neither of the tested phenols affected follicle stimulating hormone (FSH)-stimulated progesterone production, except for OP and NP at 10(-4)M, which decreased progesterone levels. The phthalates, tested at higher concentrations, were able to amplify FSH-stimulated progesterone release into the culture medium. An inhibitory action on oestradiol production by porcine granulosa cells was observed after the treatment with both groups of test chemicals. The results obtained in the experiments on primary granulosa cell cultures indicate that ovarian steroidogenesis might be one of the possible sites affected by the endocrine disrupting actions of phenols and phthalates.

Editor's Highlight: Structure-Based Investigation on the Binding and Activation of Typical Pesticides With Thyroid Receptor.

A broad range of pesticides have been reported to interfere with the normal function of the thyroid endocrine system. However, the precise mechanism(s) of action has not yet been thoroughly elucidated. In this study, 21 pesticides were assessed for their binding interactions and the potential to disrupt thyroid homeostasis. In the GH3 luciferase reporter gene assays, 5 of the pesticides tested had agonistic effects in the order of procymidone > imidacloprid > mancozeb > fluroxypyr > atrazine. 11 pesticides inhibited luciferase activity of T3 to varying degrees, demonstrating their antagonistic activity. And there are 4 pesticides showed mixed effects when treated with different concentrations. Surface plasmon resonance (SPR) biosensor technique was used to directly measure the binding interactions of these pesticides to the human thyroid hormone receptor (hTR). 13 pesticides were observed to bind directly with TR, with a KD ranging from 4.80E-08 M to 9.44E-07 M. The association and disassociation of the hTR/pesticide complex revealed 2 distinctive binding modes between the agonists and antagonists. At the same time, a different binding mode was displayed by the pesticides showed mix agonist and antagonist activity. In addition, the molecular docking simulation analyses indicated that the interaction energy calculated by CDOCKER for the agonists and antagonists correlated well with the KD values measured by the surface plasmon resonance assay. These results help to explain the differences of the TR activities of these tested pesticides.

T-screen to quantify functional potentiating, antagonistic and thyroid hormone-like activities of poly halogenated aromatic hydrocarbons (PHAHs).

The present study investigates chemical thyroid hormone disruption at the level of thyroid hormone receptor (TR) functioning. To this end the (ant)agonistic action of a series of xenobiotics was tested in the newly developed T-screen. This assay makes use of a GH3 rat pituitary cell line, that specifically proliferates when exposed to 3,3',5-triiodo-L-thyronine (T3). The growth stimulatory effect is mediated via T3-receptors. (Ant)agonistic and potentiating action of compounds was studied in absence and presence of T3 at its EC50 level (0.25 nM). The compounds tested included the specific TR-antagonist amiodarone, as well as a series of brominated diphenyl ethers (BDEs), including specifically synthesized BDEs with a structural resemblance to 3,5-diiodo-L-thyronine (T2), T3 and T4 (3,3',5,5'-tetraiodo-L-thyronine). The results obtained reveal that only BDE206 and amiodarone are specific antagonists. Interestingly some compounds which did not respond in the T-screen in absence of T3, potentiated effects when tested in combination with T3. This points at possibilities for disruption at the TR in vivo, where exposure generally occurs in presence of T3. Altogether the results of the present study show that the newly developed T-screen can be used as a valuable tool for identification and quantification of compounds active in disturbing thyroid hormone homeostasis at the level of TR-functioning.

Effects of endocrine disruptors on developmental and reproductive functions.

Endocrine disruptors (EDs) are exogenous environmental molecules that may affect the synthesis, secretion, transport, metabolism, binding, action, and catabolism of natural hormones in the body. EDs may thus interact with the endocrine system of animals and humans and can exert this effect even when present in minute amounts. EDs have adverse impacts on a number of developmental functions in wildlife and humans. Critical periods of urogenital tract and nervous system development in-utero and during early post-natal life are especially sensitive to hormonal disruption. Furthermore a wide range of hormone-dependent organs (pituitary gland, hypothalamus, reproductive tract) are targets of EDs disrupting effects in adult subjects, possibly resulting in cell transformation and cancer. At present about 60 chemicals have been identified and characterized as EDs and belong to three main groups: (a) synthetic compounds utilized in industry, agriculture and consumer products; (b) synthetic molecules used as pharmaceutical drugs and (c) natural chemicals found in human and animal food (phytoestrogens). In the present review we will give special attention to the family of Polychlorinated biphenyls (also indicated as PCBs) because of their persistence in the environment, ability to concentrate up the food chain, continued detection in environmental matrices, and ability to be stored in the adipose tissue of animals as well as humans. The detrimental effects of these compounds, and of EDs more in general, on health and reproduction will be discussed, presenting experimental data aimed at understanding the molecular mechanisms involved in their action.

Comparative study of the endocrine-disrupting activity of bisphenol A and 19 related compounds.

The endocrine-disrupting activities of bisphenol A (BPA) and 19 related compounds were comparatively examined by means of different in vitro and in vivo reporter assays. BPA and some related compounds exhibited estrogenic activity in human breast cancer cell line MCF-7, but there were remarkable differences in activity. Tetrachlorobisphenol A (TCBPA) showed the highest activity, followed by bisphenol B, BPA, and tetramethylbisphenol A (TMBPA); 2,2-bis(4-hydroxyphenyl)-1-propanol, 1,1-bis(4-hydroxyphenyl)propionic acid and 2,2-diphenylpropane showed little or no activity. Anti-estrogenic activity against 17beta-estradiol was observed with TMBPA and tetrabromobisphenol A (TBBPA). TCBPA, TBBPA, and BPA gave positive responses in the in vivo uterotrophic assay using ovariectomized mice. In contrast, BPA and some related compounds showed significant inhibitory effects on the androgenic activity of 5alpha-dihydrotestosterone in mouse fibroblast cell line NIH3T3. TMBPA showed the highest antagonistic activity, followed by bisphenol AF, bisphenol AD, bisphenol B, and BPA. However, TBBPA, TCBPA, and 2,2-diphenylpropane were inactive. TBBPA, TCBPA, TMBPA, and 3,3'-dimethylbisphenol A exhibited significant thyroid hormonal activity towards rat pituitary cell line GH3, which releases growth hormone in a thyroid hormone-dependent manner. However, BPA and other derivatives did not show such activity. The results suggest that the 4-hydroxyl group of the A-phenyl ring and the B-phenyl ring of BPA derivatives are required for these hormonal activities, and substituents at the 3,5-positions of the phenyl rings and the bridging alkyl moiety markedly influence the activities.

Subchronic exposure to low concentrations of di-n-butyl phthalate disrupts spermatogenesis in Xenopus laevis frogs.

Due to its widespread use and production, di-n-butyl phthalate (DBP) has become an environmental contaminant. It has been detected in a variety of environmental strata worldwide, including air, water, and soil. Also, monobutyl phthalate, the major metabolite of DBP, has been detected in a variety of human matrices. As a proven endocrine disruptive compound, DBP may contribute to global amphibian declines at much lower concentrations than tested thus far. We evaluated the effects of low concentrations of DBP on spermatogenesis in Xenopus laevis, the African clawed frog. Xenopus tadpoles were exposed to 0, 0.1, 0.5, 1.0, 5.0, or 10.0 ppm DBP, beginning at sexual differentiation (Nieuwkoop and Faber stage 52; 3 weeks of age) and continuing until 100% of controls metamorphosed (stage 66; 8 weeks of age). Upon necropsy at 33 weeks, 4-6% of DBP-treated frogs had only one testis, and 2-4% had retained oviducts. In all DBP treatment groups, seminiferous tubule diameter and the average number of germ cell nests per tubule were lower, and the number of tubules with no germ cells was significantly higher (p < 0.05). The percent of secondary spermatogonial cell nests significantly decreased (p < 0.05) in 1.0, 5.0, and 10.0 ppm groups. Several lesions occurred in DBP-exposed testes including denudation of germ cells, vacuolization of Sertoli cell cytoplasm, thickening of lamina propria of seminiferous tubules, and focal lymphocytic infiltration. Entire sections of testes containing almost exclusively mature spermatozoa were found in 1.0, 5.0, and 10.0 ppm DBP-exposed testes, indicating impairment of spermiation. Testicular hypoplasia and seminiferous tubular dysgenesis were also evident in DBP-treated frogs. Thus, subchronic exposure to low concentrations of DBP impairs spermatogenesis in Xenopus laevis frogs.

Detection of thyroid system-disrupting chemicals using in vitro and in vivo screening assays in Xenopus laevis.

We developed a thyroid hormone (TH) inducible primary screening assay for the identification and assessment of man-made chemicals that interfere with the TH-signalling pathway within target cells. The assay was developed in a Xenopus laevis cell line that was transduced with a self-inactivating (SIN) lentivirus vector (LV) containing a luciferase gene. The luciferase activation in this cell line was TH-specific: 3,3',5-L-triiodothyronine (T(3)) > 3,3'5-L-triiodothyroacetic acid (Triac) > 3,3',5-D-triiodothyronine (D-T(3)), > L-thyroxine (T(4)) > 3,3',5'-L-triiodothyronine (rT(3)). The application of the ligand-dependent luciferase assay for screening for thyroid system-disrupting chemicals revealed that three phthalates (dicyclohexyl phthalate, n-butylbenzyl phthalate, and di-n-butyl phthalate), two herbicides (ioxynil and pentachlorophenol) and a miticide (dicofol) had 3,3',5-L-triiodothyronine- T(3)- antagonist activity at concentrations ranging from 10(-6) to 10(-5) M. These chemicals also inhibited the expression of the endogenous primary T(3)-response TH nuclear receptor beta (TRbeta) gene. The inhibitory characteristics of these chemicals were similar for both assays performed, although the assay for T(3)-dependent activation of TRbeta gene was more sensitive than the luciferase assay. These results indicate that the luciferase assay was a rapid method with a small intra-assay variation for the primary screening of thyroid system-disrupting chemicals. Of the six chemicals, only n-butylbenzyl phthalate and pentachlorophenol exhibited T(3)-antagonist activity in an in vivo metamorphosis-based assay. It should be noted that chemicals elicited thyroid system-disrupting activity in the luciferase assay did not always interfere with the thyroid system in vivo.

Marijuana extracts possess the effects like the endocrine disrupting chemicals.

The progesterone 17alpha-hydroxylase activity, which is one of the steroidogenic enzymes in rat testis microsomes, was significantly inhibited by crude marijuana extracts from Delta(9)-tetrahydrocannabinolic acid (THCA)- and cannabidiolic acid (CBDA)-strains. Delta(9)-Tetrahydrocannabinol, cannabidiol and cannabinol also inhibited the enzymatic activity with relatively higher concentration (100-1000 microM). Testosterone 6beta- and 16alpha-hydroxylase activities together with androstenedione formation from testosterone in rat liver microsomes were also significantly inhibited by the crude marijuana extracts and the cannabinoids. Crude marijuana extracts (1 and 10 microg/ml) of THCA strain stimulated the proliferation of MCF-7 cells, although the purified cannabinoids (THC, CBD and CBN) did not show significant effects, such as the extract at the concentration of 0.01-1000 nM. These results indicate that there are some metabolic interactions between cannabinoid and steroid metabolism and that the constituents showing estrogen-like activity exist in marijuana.

A human prolactin antagonist, hPRL-G129R, inhibits breast cancer cell proliferation through induction of apoptosis.

Human breast cancer is the predominant malignancy and the leading cause of cancer death in women from Western societies. The cause of breast cancer is still unknown. Recently, the association between human prolactin (hPRL) activity and breast cancer has been reemphasized. Biologically active hPRL has been found to be produced locally by breast cancer cells that contain high levels of PRL receptor. A high incidence of mammary tumor growth has also been found in transgenic mice overexpressing lactogenic hormones. More importantly, it has been demonstrated that the receptors for sex steroids and PRL are coexpressed and cross-regulated. In this study, we report that we have designed and produced a hPRL antagonist, hPRL-G129R. By using cell proliferation assays, we have demonstrated that: (a) hPRL and E2 exhibited an additive stimulatory effect on human breast cancer cell (T-47D) proliferation; (b) hPRL-G129R possessed an inhibitory effect on T-47D cell proliferation; and (c) when antiestrogen (4-OH-tamoxifen) and anti-PRL (hPRL-G129R) agents were added together, an additive inhibitory effect was observed. We further investigated the mechanism of the inhibitory effects of hPRL-G129R in four hPRLR positive breast cancer cell lines. We report that hPRL-G129R is able to induce apoptosis in all four cell lines in a dose-dependent manner as determined by the Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay. The apoptosis is induced within 2 h of treatment at a dose as low as 50 ng/ml. We hope that the hPRL antagonist could be used to improve the outcome of human breast cancer therapy in the near future.