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It is now well established that a wide range of natural and anthropogenic chemicals present in the aquatic environment have the potential to disrupt the endocrine system of many organisms. In fish, many of these effects appear to be of a feminising nature, including stimulation of vitellogenin production and induction of intersex. In piscivorous birds these so called endocrine disrupting contaminants have been shown to impair reproduction, influencing reproductive behaviour, sex ratio, eggshell thickness and reproductive success. The effects seen in fish have been associated with high levels of oestrogenic activity in the effluent from waste water treatments works (WwTWs), but few studies have focused on the effects of WwTWs effluents on birds. In this thesis, the effects of effluents from WwTWs on fish and birds were investigated in the Tamar catchment, SW England. The work spanned making detailed assessment on the oestrogenic and anti-androgenic activity of 3 WwTWs effluents, using a variety of water sampling techniques and applying both recombinant yeast oestrogen screen (YES) and recombinant yeast androgen screen (anti-YAS) bioassays to quantify the different hormonal activities. A survey was undertaken of the hormonal activities at 13 sites to determine concentrations of contaminants in the surface waters throughout the Tamar catchment, using both recombinant yeast screens and targeted analytical chemistry for specific pollutants (LC/MS-TOF and GCMS). An ELISA was developed to quantify vitellogenin (VTG) in the bullhead (our study fish sentinel) as a biomarker of oestrogen exposure, and evidence of endocrine disruption was investigated in wild populations of the bullhead, Cottus gobio and the dipper, Cinclus cinclus. Macroinvertebrates from upstream and downstream of three WwTW's effluent discharges and from three sampling sites were also sampled as an index of overall water quality in the Tamar catchment, and as an assessment of food availability for the bullheads and dippers. For the studies on the hormonal activities in three WwTWs in the Tamar catchment, samples were collected by both spot and passive sampling; passive samplers (in replicate) were placed in the effluent discharges for a three week period, and collected on days 7, 14 and 21, spot samples were taken simultaneously. Measurement of total oestrogenic and total anti-androgenic activity was conducted using the YES and anti-YAS, respectively. Spot and passive samples were collected from 13 sites within the Tamar catchment (sampling sites were >2 km downstream of effluent discharges). Additionally, liquid chromatography mass spectrometry time-of-flight (LC/MS-TOF) was used to measure the concentration of oestrone (E1), 17β-oestrodiol (E2) and 17α-ethinylestradiol (EE2) in each sample. Gas chromatography mass spectrometry (GCMS) was used to measure the concentration of individual PBDE and PCB congeners in the spot samples only. Levels of oestrogenic and anti-androgenic activity observed in the WwTWs effluent were comparable with those measured in effluents in the UK and in other countries. Surface waters of the Tamar, away from the WwTWs effluent discharges, contained very little oestrogenic activity (<1.1 ng E2 EQs L-1), and anti-androgenic activity was undetectable. Quantification of oestrogenic activity using passive samplers showed an increasing amount of total oestrogenic activity between days 7 and 21 when measured by the both the YES and LC/MS-TOF. Low levels of PBDE congeners 47, 99, 100, 138 and 153 were detected in the spot samples taken from the Tamar catchment, with BDE 47 being the most abundant. In contrast PCBs were undetectable. Neither PBDEs nor PCBs were detected in any of the extracts from the passive samples. No assay was available to measure VTG (one of the most widely used biomarkers of oestrogen exposure in fish) in the bullhead and so an enzyme linked immunosorbant assay (ELISA) was developed for application to studies on wild bullheads in the Tamar catchment. The bullhead vitellogenin (bh-VTG) ELISA was developed successfully, and proved to be sensitive and robust, with a detection range between 10.5 and 300 ng bh-VTG mL-1 (undiluted), comparing favourably with other fish VTG ELISAs. Plasma VTG concentrations measured in male bullheads (collected from the same sites as for the water samples) ranged from below the limit of detection to 990 ng bh-VTG mL-1. Whether these upper levels in the range reflected VTG induction was difficult to conclude. Because of this controlled caged exposures with bullheads and trout were used to assess the relative levels of oestrogenicity in two key WwTWs effluent discharges and to determine the response sensitivity of the bullheads (and trout) to those effluents. These controlled exposures found no responses in plasma VTG in bullheads (ranging between 126 and 934 ng bh-VTG mL-1) suggesting a lack of sensitivity for VTG induction. This was supported by the inability to induce VTG in fish held in the laboratory and treated with steroidal oestrogens. For the effluent exposures on the caged rainbow trout, it was also found that there was no significant induction of VTG, a species normally sensitive to oestrogens. These findings may indicate that the fish were highly stressed due to the river being in spate and the movement of the cages during the controlled exposures. It may also be the case, however, that the use of immature female rainbow trout with a highly variable baseline plasma VTG concentration may prevent any detection of a response. There were no signs of sexual disruption in any of the gonads analysed from either male or female wild bullheads, demonstrating that any hormonal activity present in the catchment away from the WwTWs effluents was not sufficient to induce adverse effects on reproductive development. An interesting feature noted in the male testes of the bullheads was the presence of spermatid masses, which have been recorded in 10 other Cottidae species, but not previously in the bullhead. For the studies on dippers, eggs were collected from the nests of breeding dippers to measure for sperm numbers and morphology from sperm trapped in the perivitelline membrane (PVM), and the yolks were analysed for PBDEs, PCBs and organochlorine pesticides (OCPs) by GCMS, for E1, E2, and EE2 by LC/MS-TOF. Eggs of the dipper were collected from nests at the 13 sampling sites, plus an additional three sites and over three years of field study. The number of sperm trapped in the PVM ranged between three and 188, with a mean of 68.78 ± 8.78 SE. Dipper sperm had not previously been characterised, and was found to be similar to other passerine sperm, in that the head was helical, complemented by a mitochondrial helix or keel, which continued in a spiral around the flagellum. Sperm were classed as ‘abnormal’ if they did not adhere to this typical structure. No assessment of motility could be made in relation to the structural abnormalities seen. Contaminants in the dipper eggs were dominated by BDE 99, an unusual result considering the dippers aquatic lifestyle. PCB 153 was the most common PCB, and p,p’-DDE was the most abundant OCP; all other pesticides tested were below the limit of detection, as were the levels of all three steroid oestrogens. There was inter- and intra-nest variability between contaminant burdens in all eggs as well as the number of sperm trapped in the PVM, but there was no relationship between sperm number and the level of contaminant loadings in the eggs. There were no correlations between contaminants and oestrogenic activity measured in the water samples, and plasma VTG concentrations in bullheads or contaminant loadings in eggs, or indeed sperm number. Analysis of macroinvertebrate assemblages proved that the surface waters of the Tamar catchment were of ‘very good’ quality, even in close proximity to WwTWs effluent discharges.

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During pregnancy, increased uterine blood-flow and efficient placental perfusion is essential for a successful outcome. Despite the essential role of these vascular beds, data on the physiological mechanisms involved in the maintenance of a high-flow/low resistance circulation within the uterus and placenta are limited. The need to fully understand the regulation of blood-flow within the uterine and feto-placental circulations is further highlighted by pathological pregnancies which are characterised by vascular dysfunction within these circulations. Oestrogen and insulin-like growth factor-I (IGF-I) levels increase during pregnancy and correlate with increased uterine blood flow. In vivo and in vitro studies of other vascular beds show that both 17-β oestradiol and IGF-I act as vasodilators. However, surprisingly little is known of their vaso-active effects on human uterine and placental arteries. The aims of the studies described within this thesis, were to investigate, ex vivo, the possible roles of oestrogen and IGF-I in regulating human placental and uterine vascular beds in vivo. Placental chorionic plate arteries and myometrial demonstrated acute vasodilation in response to oestrogen. Vascular bed differences in ER-responsiveness were observed; vasodilation within myometrial arteries was elicited by both oestrogen receptors, ERα and ERβ, although activation of the latter receptor generated a greater response. In contrast, oestrogen-dependent acute vasodilation of placental arteries was via ERβ alone. Furthermore, species differences, between human and rat arteries, were demonstrated in terms of ER-responsiveness. The predominant ER receptor within human arteries studied was ERβ, whilst rat arteries demonstrated a predominantly ERα-mediated mechanism of oestrogen-induced vasodilation. The data presented suggests that within the uterine vascular bed, oestrogen-induced vasodilation involves both an endothelium-dependent and –independent mechanism of action, whilst within the placenta, oestrogen-mediated vasodilation is endothelial-independent. Indeed, data suggests that oestrogen influences the level of intracellular calcium of vascular smooth cells to induce vasodilation of placental arteries.IGF-I did not have a vaso-active effect on chorionic plate arteries isolated from the placenta. However, uterine myometrial arteries exhibited reduced vaso-reactivity in the presence of IGF-I, demonstrated by a depressed response to the vasoconstrictor, U46619. Collectively, these data contribute towards a further understanding of the regulatory mechanisms of the uterine circulation, by identifying oestrogen and IGF-I as possible regulators of the uterine vasculature during pregnancy. Additionally, oestrogen may also have a role in controlling the feto-placental circulation. In the future, targeting ERb may offer a therapeutic strategy for increasing uterine/placental perfusion in pregnancies complicated by vascular dysfunction.

Ph.D., Faculty of Health Sciences, University of the Witwatersrand, 2011 / Oestrogen receptor (ER) mutations have been identified for both ERα and ERβ in previous studies. The effects of the deletion variants due to splice mutations on clinical parameters, prognosis and treatment were examined in 61 breast carcinoma patients and 13 control samples from elective reduction mammoplasty procedures, respectively. RNA extracted from fine needle aspirates (FNAs) of breast tissue was reverse transcribed and using nested PCR and sequence analysis the presence of these variants elucidated. Using Χ2 and Fisher’s exact tests their significance with respect to clinical parameters such as tumour size, nodal involvement, stage, presence or absence of metastases, menstrual status and hormone responsiveness was examined. Kaplan-Meier survival analysis was also determined. The T-47D breast cancer cell line was cloned with two clones being selected for further analysis, namely TCA3 (hormone sensitive) and TCC1 (hormone resistant). These clones were treated for ten passages with oestrogen metabolites, 17-β- oestradiol and oestriol; oestrogen precursors, androstenedione and cholesterol; an anti-oestrogen, 4-hydroxy-tamoxifen; and the aromatase inhibitor aminoglutethimide, respectively. RNA was extracted from the cells initially and after the tenth passage and the ERα and ERβ exon profiles were examined using RT-PCR and sequence analysis. After the tenth passage hormone response tests were performed every 24 hours (up to 96 hours) with cell number being determined using the MTT assay. The results indicate that ERα and ERβ variants do not have any affect with respect to menstrual status and nodal involvement (N). Expression of ERα2 and ERα4 are required by the mouse monoclonal antibody (DAKO ® Clone 1D5) in the immunocytochemical assay used for the recognition of the protein in order to assess ER status and therefore show significance. ERαΔ2 and, contrary to previous investigations, the variant ERαΔ3 were not found to play a role in tumourigenesis. ERαΔ5 was observed to be more prevalent in ERα-positive patients and was usually co-expressed with the complete ERα5 indicating heterodimerization. ERαΔ5 showed no significance with respect to progression of disease or response to hormone treatment. An increase in the ratio of ERαΔ4: wild-type ERα4 indicated an increase in metastatic potential of diseased tissue. ERα4 and ERαΔ4 heterodimers were present in both T-47D clones and after 10 passages the TCA3 clone grown in 10-8M aminoglutethimide indicated a complete loss of ERα4 without altering hormone responsiveness. These results suggest that ERαΔ4 may play a role in progression of disease but not in the acquisition of tamoxifen resistance. ERαΔ6 was observed in 15% of patients but not in the T-47D clones or the control samples. An increase in the expression of ERαΔ6 among patient samples significantly increased their metastatic potential (p=0.018). ERαΔ6 was also observed as significant with respect to stage of disease (p=0.023) indicating the possible relevance of ERαΔ6 in progression of the disease. ERαΔ7 was the most frequently observed variant and did not show any significance with regard to any of the clinical parameters examined. The presence of ERαΔ7 did not show significance with regard to hormone response in vivo but in vitro the presence of this variant, expressed as a heterodimer with the wild-type ERα7, conferred greater sensitivity to tamoxifen in the tamoxifen resistant clone TCC1. Multiple exon deletions of ERα were also observed. The two more significant multiple deletion variants were those involving ERαΔ4, namely, ERαΔ2-ERαΔ6 and ERαΔ4-ERαΔ6. The multiple variant ERαΔ4-ERαΔ6 may be involved in tumour progression. ERβ variants were not examined in as much detail as ERα variants due to insufficient material available for analysis. The two domains, the DNA binding domain and the ligand binding domain, of ERβ were analyzed in a few of the patients and in the T-47D clones. They were not found to be significant with respect to the clinical parameters investigated and the ERβ profiles of the TCA3 and TCC1 clones remained unchanged after 10 passages under varying growth conditions.

Oestrogens are female sex hormones. Both natural and synthetic oestrogens have been found in many aquatic environments. There are three naturally occurring oestrogens – oestradiol, estriol and estrone. Oestradiol (correctly known as 17β oestradiol or E2) is a naturally occurring steroid hormone and is the most potent of these three. Diethylstilbestrol, dienestrol, quinestrol etc are synthetic oestrogens. These environmental steroidal and nonsteroidal oestrogens act as endocrine disruptors (EDs). Both types of oestrogens in the environmental samples can be quantified using several laboratory methods such as high pressure liquid chromatography (HPLC), gel permeation chromatography (GPC) etc. but they often require extensive training to perform. Arxula adeninivorans is biotechnologically significant dimorphic yeast with unusual characteristics. It can use a wide range of substrates and it is thermotolerant, osmotolerant and halotolerant. It is a non-pathogenic fungus and is therefore ideal for use in industrial settings. It is a source of many enzymes and a wide range of transformation platforms have been developed to enable the production of foreign proteins. In this project, A.adeninivorans was transformed with histidine-tagged synthetic oestrogen binding protein (EBP) gene based on the Candida albicans EBP sequence. The recombinant EBP expressed in the yeast Arxula is separated using HisTrap columns. Linear sweep voltammetry was used for the detection of EBP redox responses to oestrogen in solution. A three-electrode configuration was used for all measurements [auxiliary electrode (platinum wire), reference electrode (Ag/AgCl) and working electrodes (Pt 50μm diameter micro-disc and 2 mm diameter glassy carbon)]. Electron transfer from EBP to electrodes will require the use of a mediator system and TMPD, a lipophilic mediator used in this experiment. Screen printed electrodes (SPEs) were used to detect the interaction between EBP and oestrogen. To perform experiment with SPE, EBP was immobilized on SPE using the crosslinker glutaraldehyde. Differential pulse voltammetry (DPV) was used to detect interactions of EBP and oestrogen on SPE. Immobilion N transfer membrane was impregnated with TMPD solution and electrochemistry (DPV) was performed. The purpose of using membrane is to simulate the immobilization of TMPD on SPE along with EBP for the detection of oestrogen.

The human endometrium is a complex multicellular tissue, located within the cavity of the uterus. Its luminal surface is defined by a layer of epithelial cells supported on a multicellular stroma containing fibroblasts, glands (lined by a secretory epithelium), blood vessels (lined with endothelial cells) and several populations of immune cells; the latter includes a unique population of natural killer (uNK) cells. The endometrium undergoes dynamic remodelling across the menstrual cycle in response to fluctuating levels of sex steroids secreted by ovarian cells. The phases of the endometrial cycle include an oestrogendominated proliferative phase, a progesterone-dominated secretory phase and menses (endometrial shedding precipitated by falling levels of progesterone). A key feature of the secretory phase is differentiation (decidualisation) of endometrial stromal fibroblasts (ESC) an event characterised by transformation of cell shape, secretion of growth factors/cytokines, angiogenesis/vascular remodelling and an increase in the numbers of resident immune cells. Decidualisation ensures an appropriate nutritional and hormonal environment exists during the establishment of pregnancy. Studies in mice suggest that de novo biosynthesis of oestrogen within the uterus may play an essential role in regulation of decidualisation but no data exist for human. Endometrial endothelial and uNK cells both contain oestrogen receptors but the impact of oestrogens on their function has not been explored. In the current studies three questions have been addressed: 1. Is oestrogen biosynthesis a feature of human endometrial stromal cell decidualisation? 2. What is the impact of oestrogen on uNK cell function? 3. What role (if any) does oestrogen play in the interplay between decidual, immune and vascular cells within the human endometrial stroma? Results obtained provide the first evidence that de novo biosynthesis of oestrogens occurs during decidualisation of human ESC. This was attributed to changes in expression patterns of mRNAs encoding proteins that play a critical role in regulation of oestrogen biosynthesis (STAR, CYP11A1, CYP19A1 [aromatase], HSD17B2 [17βHSD2] and STS [steroid sulphatase]). Changes in the pattern of metabolism were confirmed using thin layer chromatography and analysis of concentrations of oestrone (E1) and oestradiol (E2) in culture media. Secretion of E1 and E2 was reduced by addition of an aromatase inhibitor. Data derived from studies described within this thesis also show for the first time that incubation of uNK cells with E2 not only enhanced cell migration but also stimulated secretion of factors that had a significant impact on endothelial cell angiogenesis. These findings were supported by novel evidence that E2 had a significant impact on expression of genes associated with cell motility and angiogenesis. In addition, factors, including E1/E2, secreted by decidualised stromal cells, stimulated chemotaxis of uNK cells. Future experiments will focus on determining the identity of the angiogenic factors secreted by uNK cells in response to E2 and the mechanisms responsible for uNK cell movement. In summary, new data presented in this thesis provide evidence that local biosynthesis of oestrogens within the endometrial stroma may play a previously unrecognised role in regulating the function of uNK cells and endometrial endothelial cells in women. These results have implications for treatment of disorders such as infertility, heavy menstrual bleeding and endometriosis.

No description available.