Search

Chemical contaminants present in the environment have the potential to negatively impact fish. This study encompasses two chemical contaminants of concern: the synthetic estrogen, 17α-ethynylestradiol (EE2) and the neonicotinoid insecticide, imidacloprid (IMI). In chapters 2 and 3, I investigate the effects of EE2 exposure on an estrogen receptor gene (esr1) in adult fathead minnows (Pimephales promelas) through evaluation of epigenetic, transcriptomic and isoform usage end points. This was accomplished by conducting a 48h exposure of adult male fathead minnows to two doses of EE2 and conducting necropsies to remove brain and liver tissues for comparison to untreated female fish. Furthermore, a subset of male fish was subjected to a 7d and 14d depuration period to evaluate the lasting effects of EE2 exposure. A targeted approach was used to evaluate DNA methylation and transcriptomic changes to esr1. I found that EE2 induced significant esr1 expression in liver tissues for both experimental groups compared to control. Targeted bisulfite sequencing revealed DNA methylation changes in both the esr1 promoter and gene body regions of exposed male fish which exhibited a female pattern. Evidence was found for some methylation marks to persist after chemical exposure, even when gene expression changes were no longer significant. Next, an RNAseq dataset was utilized to detect differential isoform usage of esr1 through comparison to reference sequences predicted by genome assembly models. The results of this study revealed significant differential isoform usage in liver tissue from male fathead minnows for esr1 transcript variants to look more similar to isoform usage found for female fathead minnows indicating that regulatory mechanisms of esr1 may play a pivotal role in the inter-sex and gonadal sex changes in fish known to occur in the presence of EE2. The study presented in chapter 4 investigates the effects of IMI exposure to zebrafish in early life stages. In this study, I generated and RNAseq dataset from zebrafish larvae exposed to IMI from 4 hours post fertilization (hpf) to 5 days post fertilization (dpf) for comparison to global DNA methylation changes resulting from reduced representation bisulfite sequencing (RRBS). I found evidence for IMI to potentially act as a circadian disruptor in zebrafish larvae through downregulation and alterations to DNA methylation of genes with roles in the circadian rhythm pathway. Behavioral differences further supported these results as IMI-exposed zebrafish larvae exhibited reduced movement during the night compared to control. Furthermore, dysregulation of neurotransmission was found to occur. Significantly reduced levels of acetylcholine, γ-aminobutyric acid (GABA), and L-glutamine (GLN) were measured in whole body larvae at the high dose of IMI. Taken together, this study provides novel insight into circadian rhythm disruption in fish in response to a neonicotinoid pesticide. Taken together, the research presented in this dissertation provides greater insight into the effects of chemical exposure on fish. Investigating the molecular mechanisms which underlie gene expression is critical to gain a broader understanding of the biological effects exerted on wildlife populations by environmental toxicants.

Circadian rhythms are approximately 24-hour cycles of behavior, physiology, and gene expression. In mammals, these circadian rhythms are generated by clocks located in nearly every tissue in the body. The function of circadian clocks is to synchronize physiology and behavior with environmental cycles such as the light-dark cycle. After menopause, when circulating levels of estrogens are very low, women are more susceptible to obesity comorbidities such as metabolic syndrome and cardiovascular disease, suggesting that estrogens regulate these processes. Estrogens could protect females from metabolic dysfunction by regulating circadian rhythms. Consumption of diets that are high in fat contribute to obesity. Consumption of high-fat diet (HFD) disrupts daily rhythms in male mice and they develop diet-induced obesity (DIO). In contrast, previous studies have shown that female C57BL/6J mice are resistant to HFD-induced obesity. Daily rhythms in female C57BL/6J mice are also resistant to disruption by HFD feeding. Female mice maintain robust daily rhythms of eating behavior and locomotor activity during HFD feeding. The circadian organization of molecular rhythms in tissues in female mice are also protected from HFD-induced disruption. When circulating estrogens are removed by ovariectomy (OVX), females are susceptible to DIO when fed HFD, just like male mice. HFD feeding also disrupts daily rhythms in OVX females. The goal of this dissertation was to investigate the role of estrogens in protecting daily rhythms in female mice from disruption by HFD feeding. We found that estradiol replacement to physiological levels in OVX females restored high-amplitude eating behavior and activity rhythms during HFD feeding so they resembled intact females. Time-restricted feeding of OVX females at the proper time of the day (12-hour active phase) inhibited obesity and improved glucose tolerance and insulin sensitivity. We next investigated the mechanism by which estradiol signaling mediates its protective effect against disruption of daily metabolic rhythms in female mice. We measured the effects of HFD feeding on daily rhythms in global estrogen receptor α knockout (ERα KO) female mice. We found that ERα was necessary to protect daily eating behavior and locomotor activity rhythms from disruption by HFD feeding. The phases of circadian clocks in tissues were also altered in ERα KO mice. The phases of PER2::LUC rhythms in the SCN and liver were advanced in both male and female ERαKO mice compared to ERα WT mice fed low-fat diet. HFD feeding exacerbated the phase advance of the live circadian clock in female ERα KO mice, but not in ERα WT mice. Taken together these results shows that estrogen signaling via the ERα receptor inhibits diet-induced obesity in part by regulating daily metabolic rhythms.

Benign prostatic hyperplasia (BPH) continues to remain a significant health problem. Steroid 5α-reductase 2 (SRD5A2) is the predominant enzyme responsible for prostatic development and growth. Our previous study demonstrated that there is an “androgenic to estrogenic switch” when SRD5A2 is absent in the prostate gland. Here we wished to identify if the expression of estrogen receptors (ER) is associated with the level of SRD5A2 in the prostate. The transcript and protein expression levels of ER was determined in prostatic specimens collected from patients who underwent transurethral resection of the prostate. Using databases GTEx (Genotype-Tissue Expression, n=100), TCGA (The Cancer Genome Atlas, n=496), and Single-cell RNA sequencing data from StrandLab.net, the expression of ER was identified and correlated to SRD5A2 in human prostate samples. Transcript and protein levels of SRD5A2, ESR1, and ESR2 in SRD5A2 transfected human prostatic stromal cell line (BHPrS1) and epithelial cell line (BPH1) were compared. ERα and ERβ were found to express variably in both the stroma and epithelium compartments. ERα was dominantly expressed in the myofibroblasts, and ERβ was mainly expressed in the endothelial cells in the prostate. ESR1 was positively correlated with SRD5A2 in human benign and malignant prostate tissues. The expression of ERα both in transcript and protein levels were promoted with the over-expression of SRD5A2 in transfected BHPrS1 cells. Overall, this study demonstrated that the expression of ER is associated with SRD5A2 in the prostate.

Liver is one of the most essential organs involved in the regulation of energy homeostasis. Hepatic steatosis, a major manifestation of metabolic syndrome, is associated with imbalance between lipid formation and breakdown and glucose production and catabolism. Although the most common risk factors of hepatocellular carcinoma (HCC) are hepatic virus infection and alcohol, a rapid increase in obesity has become a prime cause of HCC, outweighing HCC with virus- or alcohol- related etiology. Estrogens affect both cell metabolism and proliferation. Both nuclear estrogen receptors (ERs), ER-a and ER-ß, are expressed in the liver. How estrogen hormones regulate liver homeostasis and which receptors play the fundamental role in the metabolic process are not clear. Moreover, whether estrogen hormones and ERs are protective or detrimental in HCC is under debate, and whether estrogens can alter HCC metabolism and interfere with leptin-induced HCC is not known. The goal of my dissertation is to further elucidate the roles of estrogens and ERs in normal liver metabolism as well as HCC metabolism and proliferation using both in vivo and in vitro models. Thus, I hypothesize that 1) exogenous estrogen replacement reverses liver metabolic alteration in estrogen-depleted mice mainly through ER-a receptor; 2) estrogens inhibit leptin-induced HepG2 cell proliferation mainly through ER-ß activation and alter HepG2 metabolism mainly through ER-a activation. In this dissertation, previous studies on the functions of estrogen and estrogen receptors in liver and liver cancer are discussed in Chapter 1. Studies using ovariectomized mouse model with hormone-replacement was applied to determine the role of estrogens and estrogen receptors in mouse liver metabolism are discussed in Chapter 2. Human cancer cell line HepG2 was treated with different concentrations of estrogen and estrogen receptor agonists as well as estrogen receptor siRNA to determine the role of estrogen and estrogen receptors on HepG2 cancer cell proliferation, apoptosis and leptin signal pathway in Chapter 3. High performance liquid chromatography (HPLC) was used to determine the role of estrogen and estrogen receptors on HepG2 cancer cell gene expression and metabolic profiles in Chapter 4. In Chapter Five, I conclude the finding of my current studies and give perspectives for future research directions of estrogen hormones and estrogen receptors in the regulation of liver and liver cancer metabolism.

Breast cancer (BCa) is the leading cause of cancer-related death in women worldwide, while prostate cancer (PCa) is the second-leading cause of cancer-related death in men in the U.S.A. We urge to understand molecular mechanisms of cancer progression in order to develop effective treatments. Both cancers are considered to be endocrine-related cancers because sex hormones are responsible for their tumorigenesis, and there is growing evidence estrogens play an important role in promoting PCa and BCa pathogenesis. Estrogen actions are mainly mediated through estrogen receptors (ERs), which are divided into 2 subtypes, a and β1. Of these, ERβ1 is known for its protective role in PCa and BCa due to its antiproliferative and proapoptotic actions. Recently, different splice variants of ERβ1 have been discovered. Differential expression patterns and transcriptional activities of ERβ1 and its isoforms in endocrine-related cancers revealed that they possess distinct functions. The purpose of my study is to understand the mechanisms of regulation and molecular actions of ERβ1 and its isoforms. Chapter 1 gives a brief background on the development of PCa and BCa and the relationship between estrogens and their pathogenesis. The structure and molecular actions of estrogen receptors are described, while the roles of ERβ1 and its isoforms in these cancers are also introduced. Chapter 2 focuses on the regulatory mechanisms of differential expression patterns of ERβ1 and its isoforms, ERβ2 and 5, during PCa progression. Our data shows that the expression of ERβ1 is preferentially controlled by alternative promoter usage at transcriptional level, whereas that of ERβ2 and 5 is determined by complex interplay between transcriptional and post-transcriptional regulation. Chapter 3 examines the modulation of ERβ1 transcriptional activity by its coregulator, Tip60, at different DNA-binding sequences. Tip60 was found to be a dual-function coregulator of ERβ1 in a cis-regulatory element-dependent manner. It enhanced ERβ1 transactivation at activator protein 1 (AP-1) binding site but suppressed its transactivation at various estrogen response elements (EREs) in an estrogen-independent manner. Chapter 4 investigates a novel role of ERβ5 in the apoptosis of BCa cells. It was shown to confer sensitivity of BCa cells to chemotherapeutic agent (doxorubicin and cisplatin)-induced apoptosis through its interaction with a Bcl-2 family member, Bcl2L12. Chapter 5 summarizes the results, experimental limitations, future directions, significance and perspective of the studies. Also, the prospect of using ERβ1 and its isoforms as the therapeutic targets of PCa and BCa will be discussed.My thesis tries to provide more information about the molecular biology and clinical applications of ERβ I uncovered the mechanisms by which the distinct expression patterns of ERβ isoforms are regulated during PCa progression. The differential transcriptional activity of ERβ1 at different cis-regulatory sequences was shown to be determined by a dual-function coregulator. Moreover, a novel ERβ5- and Bcl2L12-mediated apoptotic pathway in BCa cells was discovered. Collectively, my data reveal that Eβ1 and its isoforms possess distinct regulatory mechanisms and signaling pathways which contribute to their unique functional roles in PCa and BCa.

Estrogens are a major group of steroid hormones that affect many systems of the body in both males and females, including the reproductive system, the urogenital tract, gastrointestinal tract, kidneys, lungs, skeletal system, immune system, cardiovascular system and the central nervous system (CNS). Within the CNS, estrogens not only play roles in the growth and maturation of neurons, neuronal excitability and synaptic connectivity, but they also affect cognition, movement, verbal fluency, performance on spatial and verbal memory tests, coordination, and fine motor skills, all of which are important for survival, finding mates, and successfully reproducing. Additionally, estrogens have been found to play important roles in regulating social behaviors when acting through estrogen receptor alpha (ER alpha), one of the two main estrogen receptors, and little is known about the effects of estrogens in the CNS when working through estrogen receptor beta (ER beta), the more recently discovered ER. The importance of ER alpha in regulating social behaviors has become a major area of research focus, especially in highly social species such as the prairie vole (Microtus ochrogaster), which exhibit the formation of pair bonds and paternal care for infants, amongst other behaviors. The role of ER beta in the CNS has since become an area of focus in highly studied animals such as lab mice and rats, but little has been done to look at its role in the highly social prairie vole. Therefore, the goal of this thesis is to localize the expression of ER beta mRNA in the prairie vole brain and compare it to expression in less social rodent species. In order to do this, in situ hybridization histochemistry was done with a radiolabeled riboprobe and autoradiography was done to view the cellular location of the message. Comparisons were done between the ER beta mRNA results of this study with ER beta-IR from a previous study on prairie voles, as well as ER beta message and protein localization data for mice and rats. ER beta mRNA was found to be in fewer areas of the prairie vole brain than the protein, and some areas of the brain where protein was not recorded in prairie voles. These differences could mean that ER beta mRNA is exported to areas of the brain where it is not made. Additionally, ER beta mRNA was found to be in fewer areas of the brain in prairie voles than in mice and rats, with the area of highest expression being the paraventricular nucleus (PVN), an area of the hypothalamus that is crucial for the production of the neurohormones, oxytocin (OT) and vasopressin (AVP), both which affect social behaviors. The differences of expression of ER beta in the prairie vole as compared to mice and rats could suggest that like ER alpha, ER beta plays roles in regulating social behaviors, and this thesis provides a background to facilitate future studies examining the roles of ER beta in regulating behaviors of highly social species.

This thesis describes medicinal chemistry methods aimed towards the understanding of estrogen receptors (ERs) and breast cancer. Breast cancer is the second most common cancer and the second leading cause of cancer fatalities in women in the United States. These devastating statistics are partially attributed to unreliable diagnosis, reoccurrence and drug resistance. Approximately 70% of these cancers express ERs, making them a relevant target for the development of new therapeutics and investigation into the mechanism of this disorder.A major portion of this project focuses on the design and synthesis of compound libraries directed towards ERs that will utilize natural products as their starting point. From a family of compounds called the flavonoids that contain estrogenic (glycinol) and anti-estrogenic (glyceollins) members, we devised new structures that are meant to exploit key binding interactions within the ER. Studies leading to these target compound libraries include the design and synthesis of `model’ compound libraries meant to mimic the target structure’s corresponding pharmacophores. By developing the model libraries we were able to explore synthetic methods that could be used for the synthesis of the targets. Additionally, one set of these libraries was also used to obtain initial biological data to develop structure-activity relationships that, in turn, contributed to final target selection.To further support our target design we studied our compound libraries (target and model) via ER docking paradigms derived from protein database starting points. To properly conduct these experiments we first determined an appropriate protein docking model for our natural product system. This docking model was designed in such a way as to best describe the biological profile of known agents and a group of our natural products. Using this protein docking model, we examined our compound libraries allowing for a more direct comparison between the two systems.Additional studies were directed toward the androgen receptor (AR) and prostate cancer. The rationale for these studies includes implications that androgens and ARs are involved in breast cancer. Furthermore, the glyceollin group from the flavonoid family has shown anti-androgen potential. Similar to the ER experiments, an X-ray derived protein docking model for the AR was devised to describe the biological profile of known agents and some of the glyceollins. This docking model was then used to study all the compound libraries.A final project was an investigation into the role of retinoic acid receptors (RARs) in breast cancer. Preliminary research implies that RARs may play a role in breast cancer drug resistance. In order to help address this hypothesis, we first synthesized a known agent to probe the RAR subtype involved in breast cancer, RARa. Secondly, we suggested other possible agents that may work as scaffolds for designing new, more specific probes. We devised a new synthetic route to these agents that ultimately led to higher yields, lower cost and decreased labor compared to that for the original standard.In this dissertation all of these topics are discussed in greater detail. Each chapter focuses on a different project and provides a synopsis of the methods utilized. Similarly, results from the methods and conclusions are given for each project including comments on their future directions.

An abstract ofDesign, Synthesis and Process Chemistry Studies ofAgents Having Anti-Cancer PropertiesbyAmarjit LuniwalSubmitted to the Graduate Faculty in partial fulfillment of the requirements for theDoctor of Philosophy Degree in Medicinal ChemistryThe University of ToledoMay 2011Breast cancer is the second leading cause of cancer deaths in American women. The use of estrogen receptor modulators is the most common treatment for early stage breast cancer and for prevention of its recurrence after surgery. However, among the available agents in this class, none display an ideal therapeutic profile. Therefore, it was aimed to design and synthesize selective estrogen receptor modulators (SERMs) which can overcome problems associated with the currently available agents. Toward that end, process chemistry studies were undertaken to enhance a synthetic route to natural glyecollin I (GLY I) which is a novel SERM that has promising anti-estrogenic and anti-cancer properties. Reaction yields across several steps were improved by optimizing reaction conditions and a few steps were improved by adopting alternative synthetic protocols. In doing so, not only were the total number of steps reduced from 15 to 13, but the overall yield was also tripled, i.e. from 3% to 9%. Molecular modeling and receptor docking studies were carried out during the design of GLY I related analogs obtained from intermediates produced during the scale-up syntheses. While performing such studies it was deduced that the active sites of the two subtypes of estrogen receptors(ERs), namely ERα and ERβ, are very similar except for two key aspects. First, the active site His residue in ERα lays slightly closer to the active site Arg and Glu as compared to the one found in ERβ. Second, the imidazole ring of the active site His residue in the two ERs is oriented quite differently in three-dimensional space. The new designed and synthesized GLY I related analogs possess pharmacophores that may be able to exploit these differences in the active sites. This, in turn, could lead to more selective estrogen receptor modulation. These analogs also possess varying degrees of flexibility while displaying their key pharmacophores important for receptor binding and selectivity.The pursuit of improved SERMs that can be derived directly from the GLY I chemistry turned next toward total synthesis of the second most prevalent member of the glyceollin family, namely GLY II. Unlike GLY I, for a specific synthesis of GLY II the 2,2-dimethylbenzopyran ring needs to be constructed first. Various chemical trials were made to accomplish this before linking two key synthons, namely 2,2-dimethylchromanone carboxylic methyl ester and an α-iodo ketone through an SN2 displacement reaction followed by an intramolecular Wittig olefination. The olefin has been instilled with the two dihydroxyl groups required for construction of a fused-benzopyran-benzofuran pterocarpan ring system. A robust, 12-step chemical route has been developed to the synthetic point of a key, late-stage intermediate which is just a few, additional steps away from the final target molecule.Model compounds were synthesized to develop and understand the chemistry associated with the key reactions utilized in the specific GLY II synthetic route. These compounds also led to the synthesis of three additional flavonoid natural products, namely 6a-hydroxymedicarpin, vestitol and bolusanthin III. Importantly, all of them possess key structural features required for binding to ERs. Therefore, they are additionally interesting for development of structure-activity relationships (SAR) that eventually can be translated into improved SERMs.Finally, analogs were similarly designed to explore SERM SAR after a new and highly practical synthetic route to a benzofuran scaffold was discovered during the specific synthesis of GLY II. The benzofuran type system represents an interesting and novel scaffold to pursue ER modulation because it is able to display polar hydroxyl groups at almost equivalent distances to those found in estradiol. These analogs were synthesized to experimentally test the two thereotical concepts pertaining to differential spacing and altered orientation of the His-imidazole ring within the ERs active site.As a second, major part of this dissertation work, the research has contributed toward the Center for Drug Design and Development‟s (CD3‟s) pursuit of chemotherapeutic treatments for advanced, hormone-independent cancer of the prostate (CaP). Similar to breast cancer in women, CaP is the second leading cause of cancer deaths in American men. Presently, there are no agents available that effectively address late-stage, advanced CaP. In this same regard, however, the CD3 previously has identified a very promising small, peptidomimetic molecule that may be able to provide some benefit in this setting. This lead compound has been designated as „CD3-246.‟ Moving forward with this technology, these thesis-related efforts involved scale-up synthetic studies and a new epimerization-free convergent route to CD3-246. In addition, an exploration of the reaction mechanism for pentamethylbenzene-mediated O-debenzylation of phenolic ethers was investigated because of its key role in the CD3-246 process chemistry. Experimental evidence was gathered to support that this reaction predominantly follows an SN1 pathway.

Tamoxifen is an effective anti-estrogen for treatment of women with hormonedependent breast cancer but acquired drug resistance limits its therapeutic benefit. We have previously reported that expression of active Akt3 in MCF-7 breast cancer cells results in estrogen-independent tumors that are actually stimulated to grow after tamoxifen treatment. We hypothesize that this tamoxifen resistance may be attributed to binding of different co-regulator proteins and/or different binding affinity of these proteins to the estrogen receptor in M CF-7 cells overexpressing active Akt3 as compared to parental MCF-7 cells. We have immuno-precipitated the estrogen receptor along with bound co-regulator proteins in both cells lines after tamoxifen, estradiol, or vehicle treatment. After 2-D gel electrophoresis separation of these immuno-precipitated proteins and comparing them using PDQuest 2-D analysis software, we identified protein spots that were statistically different under the treatment conditions between the two cell lines. The isolated protein spots were subjected to MALDI-TOF mass spectrometry. By searching protein databases through the MASCOT website for protein identification, we have identified estrogen receptor co-regulator proteins that may play a potential role in tamoxifen resistance. Current studies are focused on addressing the role of differential protein binding as a possible mechanism of tamoxifen resistance in Akt3 over-expressing breast cancer cells.

An estrogen receptor (ER) was identified in the nuclear and cytosolic fractions of the testis in the marine teleost, Atlantic croaker (Micropogonias undulatus). Competition studies demonstrated that the receptor was highly specific for estrogens and that a variety of xenoestrogens bound to the receptor with relatively low binding affinities. A comparison of the binding affinities of ligands for the testicular and hepatic ERs in this species revealed that the testicular ER saturated at a lower estradiol concentration and also had higher binding affinities for most of the estrogens and xenoestrogens tested. Minor amounts of estradiol were produced by testicular tissue fragments incubated in vitro. Incubation of testicular fragments in vitro with estradiol caused a concentration-dependent decrease in gonadotropin-stimulated 11-ketotestosterone (11-KT) production. Estradiol is acting on the cell surface via a non-genomic mechanism to alter 11-KT production