Crucial cross-talk of interleukin-1β and progesterone in human choriocarcinoma

Copyright @ 2012 Spandidos Publications Ltd. This article can be accessed from the links below.This article has been made available through the Brunel Open Access Publishing Fund.Choriocarcinoma is a highly malignant epithelial tumour that is most often associated with hydatidiform mole and presents the most common emergency medical problem in the management of trophoblast disease. We hypothesise that the hormones/cytokines present within the tumour microenvironment play key roles in the development of choriocarcinoma. In this study we assessed the effects of interleukin-1β (IL-1β) on cell death in the presence or absence of the sex hormone progesterone using two choriocarcinoma cell lines (BeWo and JEG-3) as in vitro experimental models. Although IL-1β induced cell death in both cell lines, the effect was more pronounced in JEG-3 cells, where cell death reached 40% compared to 15% in BeWo cells. Cell death of JEG-3 cells in response to IL-1β was significantly decreased by co-treatment with 100 nM and 1000 nM progesterone and completely abolished at a progesterone concentration of 1000 nM. Progesterone was also able to induce phosphorylation of ERK1/2 in these cells. Pretreatment of JEG-3 cells with a specific MAPK inhibitor (UO126) inhibited progesterone's inhibitory effect on cell death. Collectively, these data provide evidence of cross-talk between progesterone and IL-1β in this aggressive and poorly understood tumour that involves activation of a MAPK pathway and involvement of numerous progesterone receptors.This research was funded by a National Institutes of Health Grant ESO12961. This article is made available through the Brunel Open Access Publishing Fund

Characterisation and expression of β1-, β2- and β3-adrenergic receptors in the fathead minnow (Pimephales promelas)

This is the author’s version of a work that was accepted for publication in General and Comparative Endocrinology. Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. Changes may have been made to this work since it was submitted for publication. A definitive version was subsequently published and may be accessed at the link below. Copyright © 2011 Elsevier B.V. All rights reserved.Complimentary DNAs for three beta-adrenergic receptors (βARs) were isolated and characterised in the fathead minnow. The encoded proteins of 402 (β(1)AR), 397 (β(2)AR) and 434 (β(3)AR) amino acids were homologous to other vertebrate βARs, and displayed the characteristic seven transmembrane helices of G Protein-coupled receptors. Motifs and amino acids shown to be important for ligand binding were conserved in the fathead minnow receptors. Quantitative RT-PCR revealed the expression of all receptors to be highest in the heart and lowest in the ovary. However, the β(1)AR was the predominant subtype in the heart (70%), and β(3)AR the predominant subtype in the ovary (53%). In the brain, β(1)AR expression was about 200-fold higher than that of β(2)- and β(3)AR, whereas in the liver, β(2)AR expression was about 20-fold and 100-fold higher than β(3)- and β(1)AR expression, respectively. Receptor gene expression was modulated by exposure to propranolol (0.001-1mg/L) for 21days, but not in a consistent, concentration-related manner. These results show that the fathead minnow has a beta-adrenergic receptor repertoire similar to that of mammals, with the molecular signatures required for ligand binding. An exogenous ligand, the beta-blocker propranolol, is able to alter the expression profile of these receptors, although the functional relevance of such changes remains to be determined. Characterisation of the molecular targets for beta-blockers in fish will aid informed environmental risk assessments of these drugs, which are known to be present in the aquatic environment.European Union as part of the ERAPharm project, Contract No. 511135 and NER

Expression of membrane and nuclear progesterone receptors in two human placental choriocarcinoma cell lines (JEG-3 and BeWo): Effects of syncytialization

This article has been made available through the Brunel Open Access Publishing Fund and is available from the specified link - Copyright @ 2011 Spandidos Publications Ltd.A vital function of the human placenta is to produce steroid hormones such as progesterone, which are essential for the maintenance of pregnancy and the onset of parturition. Although choriocarcinoma cell lines are valuable placental models for investigations of steroid hormone actions, little is known about the expression of progesterone receptors (PRs) in these cell lines. Therefore, in this study, the expression of membrane and nuclear PRs was investigated in cultures of fusigenic (BeWo) and non-fusigenic (JEG-3) human choriocarcinoma cell lines. In addition, the effects of an inducer of syncytialization (forskolin) on the PR expression in BeWo cells were assessed. Quantitative RT-PCR revealed that in fully syncytialized BeWo cells (treated with 50 mu M forskolin for 72 h) there was a significant down-regulation of mPR alpha and up-regulation of mPR beta and of the progesterone membrane component-1 (PGRMC1) when compared with non-syncytialized BeWo cells. Expression of all the mPR and PGRMC1 mRNAs was significantly lower in JEG-3 cells compared to non-syncytialized BeWo cells. Interestingly, expression of PR-B was unaltered between the two BeWo states but was significantly higher in JEG-3 cells. Immunofluorescence analysis revealed that mPR proteins are differentially expressed in these choriocarcinoma cell lines as well as in the human placenta. The data demonstrate that human choriocarcinoma cell lines have a complex system of progesterone signalling involving multiple classes of PRs. The finding that syncytialization is accompanied by changes in the expression of these receptors may suggest that this process influences progesterone signalling

Overwinter fasting and re-feeding in rainbow trout: plasma growth hormone and cortisol levels in relation to energy mobilization

This study investigated the roles of cortisol and growth hormone during a period of fasting in overwintering salmonid fish. Indices of carbohydrate (plasma glucose, liver glycogen), lipid (plasma free fatty acids) and protein metabolism (plasma protein, total plasma amino acids) were determined, together with plasma growth hormone (GH), cortisol and somatolactin levels (SL) at intervals in three groups of rainbow trout (continuously fed; fasted for 9 weeks then fed; fasted for 17 weeks). In fasted fish, a decline in body weight and condition factor was accompanied by reduced plasma glucose and hepatic glycogen and increased plasma FFA. No consistent elevation of plasma GH occurred until after 8 weeks of fasting when plasma GH levels increased nine-fold. No changes were observed in plasma total protein and AA until between weeks 13 and 17 when both were reduced significantly. When previously fasted fish resumed feeding, plasma glucose and FFA, and hepatic glycogen levels rapidly returned to control values and weight gain resumed. No significant changes in plasma cortisol levels, related to feeding regime, were evident at any point during the study and there was no evidence that SL played an active role in the response to fasting. The results suggest that overwinter fasting may not represent a significant nutritional stressor to rainbow trout and that energy mobilisation during fasting may be achieved without the involvement of GH, cortisol or SL

Examining the suitability of molecular and metabolomic-based techniques as tools for assessing the effects of pharmaceuticals in the aquatic environment

Pharmaceuticals represent important and indispensable elements in modern society and their usage is considerable. Post consumption and body-elimination, pharmaceuticals are not completely removed in sewage treatment works (STWs) and as such, have been detected at low levels in STW effluents, surface waters, seawaters, ground waters and some drinking waters. Accordingly, pharmaceutical toxicity has been detected in several aquatic organisms. To date, environmental risk assessments (ERA) examine for toxicity using a series of chronic toxicity assays that examine for standard physiological responses in algae, Daphnia and fish and do not address pharmaceutical mode of action. Therefore, using the fathead minnow (Pimephales promelas) and the β-blocking pharmaceutical propranolol as the test-species and test-drug, respectively, the aim of this study was to establish an intelligent targeted 4-phased ERA using molecular, in vivo exposure, metabolomic and quantitative expression analytical techniques. The first phase established that the fathead minnow expressed the β3bi-adrenergic receptor (AR), which is a target receptor for propranolol in humans. The in vivo pair-breeding assay suggested that at 1mgL-1 and 10mgL-1, propranolol levels in fish blood plasma exceeded the human therapeutic concentration and caused 80% and 100% mortality, respectively. The most likely causes of mortality were liver failure and central nervous system toxicity. It was not possible to identify a robust biomarker of propranolol exposure using proton nuclear magnetic resonance (1H NMR) as there was considerable metabolic variation between male liver tissues within the same treatment groups. β3bi¬¬-AR expression was significantly lower at 1mgL-1 in the brain and liver, which was most likely the result of desensitisation in response to elevated levels of epinephrine and cortisol. β3bi¬¬-AR expression was significantly increased in the heart at the environmentally relevant concentration of 0.001mgL 1, however it was not possible to link β3bi¬¬-AR expression to a toxic response. Propranolol is unlikely to pose a threat to the aquatic environment as the concentrations measured in the environment are approximately 1000-fold lower than those that induced a toxic response. The proposed ERA represents a marked improvement over the existing ERA as it addresses pharmaceutical mode of action and both subtle and physiological toxicity responses, however it still requires further validation studies to address both metabolomic and gene expression variation.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Pharmaceuticals in the aquatic environment : β-blockers as a case study

The presence of many human pharmaceuticals in the aquatic environment is now a worldwide concern and yet little is known of the chronic effects that these bioactive substances may be having on aquatic organisms. This study used mammalian pharmacodynamics to predict the mode of action of the 13-blocker, propranolol, on fish, in order to identify chronic effects in fathead minnows. β-blockers target β1- and β2-adrenergic receptors in humans and hence these receptors were characterised in the fathead minnow. It was found that fish possess β1- and β2-ARs that are structurally very similar to their mammalian counterparts. Further, the distributions of these two β-ARs in various organs of the fathead minnow were similar to those in mammals. Pair-breeding assays were conducted, in which fathead minnows were exposed to various concentrations of propranolol. To discover whether β-ARs had been up or down regulated by propranolol, molecular analysis was conducted on different tissues of the exposed fish using real-time polymerase-chain reactions (RT-PCR). Exposure of fathead minnows to propranolol caused acute toxicity at 10 mg/L. Propranolol caused a statistically significant decrease in reproduction at 1.0 mg/L, dose-related decreases in male weight, condition index and fatpad weight, and a dose-related increase in female GSI. Molecular analysis of βl- and β2-AR expression levels revealed a dose-related decrease in β2-AR expression in fathead liver and heart. LOEC and NOEC values were 0.1 mg/L and 0.01 mg/L, respectively. Propranolol plasma concentrations in fish exposed to water concentrations of 0.1 and 1.0 mg/L were greater than the human therapeutic concentration and hence these data very strongly support the fish plasma model proposed by Huggett et al. (2001).EThOS - Electronic Theses Online ServiceEuropean Union (as part of the ERAPharm consortium)GBUnited Kingdo

Progesterone receptors in the human placenta : expression, signalling characteristics and functional relevance

The human placenta is a transient life sustaining organ which is responsible for mediating all the physiological exchanges between the mother and the fetus. The steroid hormone progesterone, often referred to as the hormone of pregnancy, is critical for the establishment and for maintaining the pregnancy. During the gestation period the human placenta produces progesterone which via interacting with the progesterone receptors exerts its many effects. Specific intracellular progesterone receptors (PRs) have been reported to mediate the genomic signalling of progesterone whereas recently two novel receptor families which are phylogenetically distinct to the nuclear receptor superfamily have been characterised, and shown to mediate progesterone’s non genomic actions. These are the multiple membrane progestin receptors (mPRα, mPRβ, and mPRγ) and progesterone membrane receptor component 1 (PGMRC-1). The rapid progesterone actions mediated via these non-classical progesterone receptors have received attention with main focus on their reproductive functions. Our aim is to elucidate the expression of the receptors in the human placenta, further understand the signalling pathways via which progesterone mediates its effects and lastly examine the functional relevance of these receptors in this organ. Choriocarcinoma cell lines are used frequently as placental models for investigations of steroid hormone actions, but until now little is known about the expression of progesterone receptors (PRs) in these cell lines. Quantitative RT-PCR revealed that in fully syncytialized BeWo cells (treated with 50 µM forskolin for 72 h) there was a significant down-regulation of mPRα and up-regulation of mPRβ and of the PGRMC1 when compared with non-syncytialized BeWo cells. Expression of all the mPR and PGRMC1 mRNAs was significantly lower in JEG-3 cells compared to non-syncytialized BeWo cells. Expression of PR-B was unaltered between the two BeWo states but was significantly higher in JEG-3 cells. Immunofluorescence analysis revealed that mPR proteins are differentially expressed in these choriocarcinoma cell lines as well as in the human placenta. The functionality of mPRs was investigated in vitro, using BeWo and JEG-3 cells that were treated with Org OD-02 (a specific mPR agonist), progesterone (P4) and R5020 (a specific nuclear PR agonist) in the presence or absence of the pro-inflammatory cytokine inteleukin-1β (IL-1β) at a concentration of 10ng/μl. The effect was more exacerbated in JEG-3 cells, where IL-1β induced 40% cell death when compared to BeWo cells that reached a modest but significant 15% cell death. When JEG-3 cells were treated with IL-1β and progesterone, there was a significant decrease in cell death at concentrations of 100nM and 1000nM. When cells were treated with 1000nM progesterone, IL-1β’s effect was completely abolished. Progesterone was also able to induce phosphorylation of ERK1/2 in these cells. Pretreatment of JEG-3 cells with a specific MAPK inhibitor (UO126) inhibited substantially the progesterone’s proliferative effect. Moreover, using the specific mPR agonist Org OD 02-0, we have shown that that the progestin antagonism of apoptotic effects of IL-1β on BeWo cells is mediated through mPRs. Quantitative PCR in clinical samples revealed a 2.8 fold decrease of mPRβ in labouring comparing to non-labouring tissues and 4.6 fold higher levels of mPRγ in preterm mPRγ compared to term placentas. The ratio of mPRα to PR-B was increased in term compared to preterm samples, whereas it was decreased in labour compared to non-labour placentas. There was also a high correlation between mPRα and PGRMC1 expression irrespective of pathologies. This study addressed many fundamental questions regarding how progestins exert their effect at placental level. It is evident that there is a higher order of complexity and changes in the ratios of placental progesterone receptors rather than individual fluctuations might affect subsequent signalling events.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Testing the translational power of the zebrafish: an inter-species analysis of responses to cardiovascular drugs

The zebrafish is rapidly emerging as a promising alternative in vivo model for the detection of drug-induced cardiovascular effects. Despite its increasing popularity, the ability of this model to inform the drug development process is often limited by the uncertainties around the quantitative relevance of zebrafish responses compared with non-clinical mammalian species and ultimately humans. Here we provide a comparative quantitative analysis of the in vivo cardiovascular responses of zebrafish, rat, dog, and human to three model compounds (propranolol, losartan, and captopril), which act as modulators of two key systems (beta-adrenergic and renin-angiotensin systems) involved in the regulation of cardiovascular functions. We used in vivo imaging techniques to generate novel experimental data of drug-mediated cardiovascular effects in zebrafish larvae. This data was combined with a database of inter-species mammalian responses (i.e. heart rate, blood flow, vessel diameter, stroke volume) extracted from the literature to perform a meta-analysis of effect size and direction across multiple species. In spite of the high heterogeneity of study design parameters, our analysis highlighted that zebrafish and human responses were largely comparable in >80% of drug/endpoint combinations. However, it also revealed a high intra-species variability which, in some cases, prevented a conclusive interpretation of the drug-induced effect. The meta-analysis approach, combined with a suitable data visualization strategy, enabled us to observe of patterns of response that would likely remain undetected with more traditional methods of qualitative comparative analysis. We propose that expanding this approach to larger datasets encompassing multiple drugs and modes-of-action, would enable a rigorous and systematic assessment of the applicability domain of the zebrafish from both a mechanistic and phenotypic standpoint. This will increase the confidence in its application for the early detection of adverse drug reactions in any major organ system.Biotechnology and Biological Sciences Research Counci

The effect of online and in-person team-based learning (TBL) on undergraduate endocrinology teaching during COVID-19 pandemic

Availability of data and materials: The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.Copyright © The Author(s) 2022. Background: Team-based learning (TBL) combines active and collaborative learning, while incorporating aspects of the flipped classroom approach and problem-based learning. The COVID-19 pandemic presented certain challenges in the delivery of TBL in class. In this study, we investigated the impact of TBL on the academic performance of final year Biomedical Sciences’ undergraduate students in the context of an “Endocrine Disorders” study block. We did so by comparing the classical in-person approach and online delivery due to the COVID-19 pandemic. Methods: A non-compulsory TBL session was introduced to the curriculum of this block, which followed the traditional 2-h lecture delivery. Comparative analysis was performed for the exam and coursework performance of students who attended the TBL sessions (online and in-person) and those that did not. Results: Both cohorts of students who attended either in-person (n = 66) or online TBL sessions (n = 109) performed significantly better in their exams (p < 0.05) and a related coursework (p < 0.001 and p < 0.05, respectively) when compared to those that did not attend. For both these cohorts the exam mark distribution was much narrower compared to those that did not attend the TBL sessions where the majority of fails and “no shows” were recorded. Conclusions: Online and in-person TBL, can successfully supplement traditional lecture-based teaching and enhance the learning/performance, for complex medical subjects/topics. Our findings demonstrate that it is possible to deliver these sessions online with demonstrable benefit for students suggesting that there is greater flexibility in the use of TBL in higher education

Investigation into the effects of pesticides on amphibians

Amphibian population decline is a recognised phenomenon spanning at least the last 40 years, and it is likely that a number of factors have contributed, including environmental contamination. Amphibians are vulnerable to agrochemical uptake as they must breed in water, and often spend the aquatic phase of their lifecycle in agricultural water bodies, which may contain a complex mixture of biologically active chemicals. Endocrine disrupting compounds may cause reproductive effects in humans and wildlife, although the link between pesticides and endocrine disruption is largely unknown. Therefore, in this study, the role of pesticides in endocrine disruption, in relation to amphibian metamorphosis and reproductive development, was investigated. To achieve this objective, population data were used to select suitable field sites, water from which was tested for endocrine activity using the yeast estrogen/androgen screen, hepatocyte culture (estrogenic response), and a transgenic Xenopus test (thyroid disruption). Toad (Bufo bufo) specimens from a subsample of these sites were used to compare morphology, thyroidal, and gonadal development of caged and wild-caught tadpoles/metamorphs, to their laboratory-raised counterparts. In addition, environmentally relevant pesticides were tested for endocrine effects in vitro, and a short-term in vivo exposure was used to assess the predictive ability of the in vitro screens in Xenopus. Mortality of Bufo bufo was high in both laboratory-reared and caged individuals, which hindered the interpretation of results due to low n values. However, laboratory-reared individuals from different sites had distinct morphology and gonadal differentiation, possibly suggesting maternal transfer, a latent effect of the pond environment, and/or genetic effects. In addition, caged and wild-caught individuals were smaller, metamorphosed later, and had retarded gonadal differentiation or increased incidence of intersex, compared to their laboratory-reared counterparts. Extracts of water samples from these sites were predominantly anti-estrogenic, and/or anti-androgenic in yeast based assays, and this was also the effect observed in response to environmentally relevant pesticides tested in the same assays. Pesticides also affected ovarian steroidogenesis in vitro, and pentachlorophenol had a reprotoxic effect on adult female Xenopus laevis. Data reported in this study suggest there may be endocrine disrupting effects in native amphibians in the agricultural landscape, although further investigation is needed to confirm these findings.EThOS - Electronic Theses Online ServiceGBUnited Kingdo