Sudhansu K. Dey - US grants

The aim of our proposed research is to investigate the physiological function of histamine and its mode of action in embryonic development and implantation. We have recently shown that the mammalian preimplantaton embryo has histamine forming capacity and that inerference with this function interrupts embryo development and implantation. Still unanswered is how histamine could be involved in these processes. On the basis of our findings and exploitation of the findings of other investigators, we propose that histamine produced by the embryo or released from the uterus by estrogen, stimulates phospholipase A2 activity, considered by many as the rate-limiting enzyme, which makes available free arachidonic acid for prostaglandin synthesis in the embryo and/or in the endometrium at the site of implantation. Histamine or prostaglandins, or both, in a cascade manner, then participate in embryo development and implantation. Radioimmunoassays and isotopic enzymatic assays will be used to study the formation of prostaglandins and histamine in the embryo and the uterus under different physiological and experimental conditions. The culture and transplantation of embryos will be used to answer physiological implications of histamine in embryogenesis and implantation. Progress in the development of novel methods of fertility regulation as well as in the area of control and prevention of abnormal development and birth defects will be advanced by information about early stages of embryogenesis and implantation. The proposed project is aimed at obtaining such information.

The production and metabolism of steroid hormones by the mammalian preimplantation embryo and their physiological significance in implantation are presently controversial. The objectives of the proposed research are aimed at these two fundamental concepts. We have recently shown that rabbit blastocysts have considerable aromatase activity (estrogen forming capacity). However, only 28% of this activity is expressed as estradiol-17Beta (E2), suggesting formation of other estrogens. One such candidate could be catechol estrogen. Recent reports indicate that catechol estrogens have uterotrophic as well as positive and negative feedback effects upon the hypothalamic-hypophyseal axis and are more potent than E2 in stimulating prostaglandin (PG) synthesis in the rat uterus in vitro. PGs are considered to participate in implantation. Therefore, we propose to investigate whether primary estrogens, can be converted to catechol estrogens in the embryo and/or the endometrium and whether these catechol estrogens can stimulate PG synthesis in the embryo and/or the endometrium. Experimental methods to be employed include culture of embryos, biochemical assays of enzymes and radioimmunoassay of PGs.

The reproductive system is unique among the bodily systems in the complexity of the mechanisms that control it. Many classes of psychoactive drugs can disrupt aspects of these controlling mechanisms and alter reproductive function. Research on the reproductive effects of drugs has numerous methodologic and interpretive problems, and several important questions remain unanswered (Reviewed by Smith & Asch, 1987). One of the most controversial issues in the research of marihuana on reproduction has been whether this drug is estrogenic or not. In the adult uterus, a large spectrum of estrogenic responses is elicited by a variety of steroidal and non-steroidal compounds. The type(s) of response(s) and the cell-types involved are dependent upon the nature of the compound and the hormonal environment the uterus is exposed to. In this respect, progesterone remarkably alters estrogenic responses in the uterus in a cell type- specific manner. Therefore, sensitive and defined systems are essential to determine the estrogenicity of marihuana, and the problem necessitates a concerted effort from various angles. The diverse but combined expertise of the investigators present a unique opportunity to fulfill this purpose. Our specific aims are to: 1) determine the stage specific effects of delta-9-tetrahydrocannabinol (THC) on preimplantation embryo development, implantation and decidualization; 2) determine if the effects of THC are on the embryo, the uterus or both; 3) determine whether THC functions as an estrogen or an estrogen antagonist in inducing implantation in delayed implanting mice; 4) determine whether the effects of THC follow phasic estrogenic responses in the ovariectomized uterus and are modulated by progesterone; 5) determine whether THC discordantly alters the gene expression (as an estrogen) induces gene expression in the ovariectomized uterus in a cell type-specific manner that is altered by progesterone pretreatment. To determine stage and site specific effects of THC during early pregnancy, the blue dye method, embryo manipulation, culture and transfer will be employed. To determine estrogenicity of THC, the model of delayed implantation, assessment of Phase I (125I-BSA uptake) and Phase II (3H-thymidine incorporation) estrogenic responses and studies of gene expression in the uterus will be used. Expression of c-myc and IGF-I genes will be determined by detection of mRNAs by northern blot hybridization and their cell type-specific expression by in situ hybridization. Immunocytochemistry will be used to localize cell type-specific accumulation of c-myc and IGF-I proteins. The results obtained from the proposed study will not only establish whether THC elicits any estrogenic effects and interferes with early pregnancy, but will also show for the first time whether THC has any effect on gene expression in the uterus. Because in most cases pregnancy failure occurs during the peri-implantation period, the proposed experiments on the effects of THC during early pregnancy will provide important information as to the cause (embryo, uterus or both?) of pregnancy failure following exposure to the drug.

DESCRIPTION: (Applicant's Abstract) The reported adverse effects of marijuana use, its growing availability and consumption in recent years have heightened public concern. The national debate between those pressuring for its legalization and those supporting its ban will continue until the adverse and beneficial effects of this drug are clearly defined. One of the major concerns, among many central and peripheral effects of habitual marijuana smoking or exposure to cannabinoid derivatives, is their reported adverse effects on reproductive functions, including retard embryonic growth, pregnancy failure and poor fertilizing capacity of sperm. The mechanism(s) by which these cannabinoid effects are mediated are poorly defined. Recent identification and cloning of G-protein coupled cannabinoid receptors in the brain (CB1-R) and spleen (CB2-R) provide evidence that many of these effects are mediated via these receptors. Further, a putative endogenous cannabinoid ligand, arachidonoylethanolamide (anandamide) has been detected in the brain. Our recent finding of expression of cannabinoid receptors in the preimplantation mouse embryo and uterus, as well as anandamide synthesizing capacity in the uterus and oviduct also place these tissues as targets for cannabinoid ligand-receptor signaling and prompted us to continue investigation on this signaling pathway during early pregnancy. Our specific aims are to determine in the mouse: (1) Expression and regulation of the CB1-R and CB2-R genes in the embryo and uterus during early pregnancy (days 1-8), (2) Anandamide synthesizing and (3) Anandamide hydrolyzing capacities, and their regulation in the oviduct and uterus during this period and under steroid hormonal stimulation, (4) Status of phospholipase A2 and phospholipase D activities in these tissues under these conditions, and (5) Roles of cannabinoid-ligand receptor signaling in the embryo and uterus during early pregnancy. We will use multiple experimental approaches including reverse transcription-polymerase chain reaction (RT-PCR),l Northern blot and in situ hybridization, immunostaining, Western blotting, autoradiographic and Scatchard analysis of ligand binding, enzyme assays, embryo culture and transfer, and others to accomplish our goal. Since there are similarities between mouse and human preimplantation and implantation physiology, results of this investigation will further our knowledge that should be valuable for defining marijuana's role in women's health issues.

9815819
Terranova

This award supports a three year collaborative research project between Professor Paul Terranova of the University of Kansas Medical Center and Professor Kazuyoshi Taya of the Tokyo University of Agriculture and Technology in Japan. The researchers will be undertaking a study of the effects of environmental agents on reproductive processes. The researchers plan to analyze the effects of several environmentally relevant toxic compounds on various aspects of reproductive function. They hope to provide new insights into the mechanisms by which reproductive function is compromised. The problems to be explored are: 1) the effects of the environmental contaminant, dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin), on ovarian function and early pregnancy; and 2) the effects of xenoestrogens [chlordecone (Kepone), and O-P'-DDT], and a natural estrogen, estradiol, on uterine gene expression of immediate early protooncogenes and several relevant uterine growth factors such as heparin binding EGF-like growth factor and lactoferrin.

This project brings together the efforts of two laboratories that have complementary expertise and research capabilities. The U.S. researchers' expertise is in the area of molecular methods of signal transduction and the Japanese have expertise in the endocrinological area. Results of this research would provide a significant advancement to the field of endocrine disruptors. This research advances international human resources through the participation of a younger scientist and graduate students. Through the exchange of ideas and technology, this project will broaden our base of basic knowledge and promote international understanding and cooperation.
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DESCRIPTION: (Applicant's Abstract) The reported adverse effects of marijuana use, its growing availability and consumption in recent years have heightened public concern. The national debate between those pressuring for its legalization and those supporting its ban will continue until the adverse and beneficial effects of this drug are clearly defined. One of the major concerns, among many central and peripheral effects of habitual marijuana smoking or exposure to cannabinoid derivatives, is their reported adverse effects on reproductive functions, including retard embryonic growth, pregnancy failure and poor fertilizing capacity of sperm. The mechanism(s) by which these cannabinoid effects are mediated are poorly defined. Recent identification and cloning of G-protein coupled cannabinoid receptors in the brain (CB1-R) and spleen (CB2-R) provide evidence that many of these effects are mediated via these receptors. Further, a putative endogenous cannabinoid ligand, arachidonoylethanolamide (anandamide) has been detected in the brain. Our recent finding of expression of cannabinoid receptors in the preimplantation mouse embryo and uterus, as well as anandamide synthesizing capacity in the uterus and oviduct also place these tissues as targets for cannabinoid ligand-receptor signaling and prompted us to continue investigation on this signaling pathway during early pregnancy. Our specific aims are to determine in the mouse: (1) Expression and regulation of the CB1-R and CB2-R genes in the embryo and uterus during early pregnancy (days 1-8), (2) Anandamide synthesizing and (3) Anandamide hydrolyzing capacities, and their regulation in the oviduct and uterus during this period and under steroid hormonal stimulation, (4) Status of phospholipase A2 and phospholipase D activities in these tissues under these conditions, and (5) Roles of cannabinoid-ligand receptor signaling in the embryo and uterus during early pregnancy. We will use multiple experimental approaches including reverse transcription-polymerase chain reaction (RT-PCR),l Northern blot and in situ hybridization, immunostaining, Western blotting, autoradiographic and Scatchard analysis of ligand binding, enzyme assays, embryo culture and transfer, and others to accomplish our goal. Since there are similarities between mouse and human preimplantation and implantation physiology, results of this investigation will further our knowledge that should be valuable for defining marijuana's role in women's health issues.

Ovarian cancer represents an impending candidate for intense research and a target for chemoprevention, because it is the most lethal gynecological malignancy and associated with a high mortality rate. The underlying causes of ovarian cancers remain elusive and treatment options for patients with advanced disease are still inadequate. While several studies suggest that the use of non-steroidal anti-inflammatory drugs (NSAIDs) is associated with a decreased risk of developing ovarian cancer, other studies failed to detect any significant association. The cyclooxygenase (COX) enzymes, COX-1 and COX-2, catalyze prostaglandin (PG) biosynthesis. Research primarily on colorectal cancer has established that NSAIDs are effective in cancer prevention, and treatment of established tumors. These drugs are believed to inhibit cancer growth primarily by inhibiting COX-2. COX-2 is also upregulated in a range of extra-colonic cancers and selective COX-2 inhibitors show potent anti-neoplastic effects. These findings have led to the initiation of several clinical trials testing the efficacy of COX-2 selective inhibitors in the prevention of cancer or as part of a combination therapy for established tumors. The expression pattern of COX isoforms in ovarian cancer remains conflicting. Using multiple approaches, our preliminary results provide evidence that both human and mouse epithelial ovarian tumors show heightened expression of COX-1, not COX-2. To define whether the COX-derived PGs play any role in ovarian cancer and to determine whether a scientific rationale exists for the use of COX inhibitors in the prevention and/or treatment of the disease, in-depth studies are warranted. We hypothesize that COX-1 derived PGs play a major role in the genesis and progression of ovarian epithelial cancer by upregulating proangiogenic events. We will test our hypothesis by using human ovarian cancer samples with or without cytoreductive treatment, human ovarian cancer cell lines and mouse models of ovarian cancers. Our specific aims are to: (1) Characterize models of ovarian cancer with respect to PG biosynthesis as well as the potential mechanisms of actions of the dominant PGs formed in executing proneoplastic effects and (2) Analyze the effects of COX inhibitors on the initiation and progression of ovarian tumors in mouse models. Our proposed genetic and molecular approaches in vivo and in vitro using human and mice as model systems will provide valuable information for better understanding of prevention and treatment of ovarian cancers.

[unreadable] DESCRIPTION (provided by applicant): The primary objective of the proposed training program in reproductive biology (RBTP) is to continue offering predoctoral and postdoctoral training to individuals who demonstrate potential for developing into independent investigators in the field of reproductive biology. The renewal application by including multidisciplinary interests expands the horizon of training for students. This will better prepare their career paths in the 21st century and present them with opportunities to make significant scientific contribution to reproductive biology. This renewal application focuses on the current excitement in scientific innovations, opportunities and advances in biomedical sciences with evolving technologies. The proposed RBTP has been developed within the current mission of the NIH roadmap to multidisciplinary approaches to research endeavors. This mission can best be accomplished if students are trained in an environment where specific biological problems are addressed using contemporary knowledge in developmental biology, physiological genomics, proteomics, signal transduction, and molecular and cellular biology. The present environment at Vanderbilt is highly conducive to such training due to a broad based interactive group of 13 investigators spanning several departments and thematic areas, and the recent establishment of genomics, proteomics, lipidomics and imaging centers on the campus with state-of-the-art facilities. Training will occur with major emphasis in: 1) gametogenesis, sperm functions and fertilization, (Greenstein, Olson, Drummond-Barbosa and Waterman); 2) uterine biology (Das, Dey, Ong and Paria); 3) embryogenesis, development and implantation (Das, Dey, Chiang, Lee and Paria); and 4) genetic regulation of gonadotropin secretion (Phillips). These areas of research training require the infusion and active participation of preceptors who are experts in developmental biology, signal transductions, genomics and proteomics (Lee, Caprioli, and Sealy). [unreadable] [unreadable] [unreadable]

Ovarian cancer represents an impending candidate for intense research and a target for chemoprevention, because it is the most lethal gynecological malignancy and associated with a high mortality rate. The underlying causes of ovarian cancers remain elusive and treatment options for patients with advanced disease are still inadequate. While several studies suggest that the use of non-steroidal anti-inflammatory drugs (NSAIDs) is associated with a decreased risk of developing ovarian cancer, other studies failed to detect any significant association. The cyclooxygenase (COX) enzymes, COX-1 and COX-2, catalyze prostaglandin (PG) biosynthesis. Research primarily on colorectal cancer has established that NSAIDs are effective in cancer prevention, and treatment of established tumors. These drugs are believed to inhibit cancer growth primarily by inhibiting COX-2. COX-2 is also upregulated in a range of extra-colonic cancers and selective COX-2 inhibitors show potent anti-neoplastic effects. These findings have led to the initiation of several clinical trials testing the efficacy of COX-2 selective inhibitors in the prevention of cancer or as part of a combination therapy for established tumors. The expression pattern of COX isoforms in ovarian cancer remains conflicting. Using multiple approaches, our preliminary results provide evidence that both human and mouse epithelial ovarian tumors show heightened expression of COX-1, not COX-2. To define whether the COX-derived PGs play any role in ovarian cancer and to determine whether a scientific rationale exists for the use of COX inhibitors in the prevention and/or treatment of the disease, in-depth studies are warranted. We hypothesize that COX-1 derived PGs play a major role in the genesis and progression of ovarian epithelial cancer by upregulating proangiogenic events. We will test our hypothesis by using human ovarian cancer samples with or without cytoreductive treatment, human ovarian cancer cell lines and mouse models of ovarian cancers. Our specific aims are to: (1) Characterize models of ovarian cancer with respect to PG biosynthesis as well as the potential mechanisms of actions of the dominant PGs formed in executing proneoplastic effects and (2) Analyze the effects of COX inhibitors on the initiation and progression of ovarian tumors in mouse models. Our proposed genetic and molecular approaches in vivo and in vitro using human and mice as model systems will provide valuable information for better understanding of prevention and treatment of ovarian cancers.

DESCRIPTION (provided by applic) Over the past two decades, it has been increasingly recognized that the risk of adult health disorders can be markedly influenced by prenatal and infant environmental and pharmaceutical exposures. The New York Academy of Sciences (NYAS) and Cincinnati Children's Hospital Medical Center are jointly presenting a 1.5- day scientific symposium to explore recent discoveries, challenges, and future research directions that further our understanding of the complex environmental and genetic factors, and gene-gene and gene- environment interactions responsible for fetal programming in utero and pre-term birth. Organized by Drs. Sudhansu K. Dey, Jeffrey A. Whitsett, and Susan J. Fisher (investigators), this event will take place at the New York Academy of Sciences conference center in New York City on June 7-8, 2012. The goals of the symposium are to: (i) discuss genetic and epigenetic factors, in addition to environmental exposures in the intrauterine environment that have a profound effect on the trajectory of prenatal development and pre-term birth, including the effects on: implantation; placentation; organogenesis; and neonatal health, and how to combat these effects; (ii) foster and highlight the research contributions of young and minority investigators working in this field; and (iii) develop high-quality, open-access, web-based enduring materials, to disseminate the latest scientific findings from this fast-moving field to the global community. The conference agenda features a Keynote Address, plenary lectures with audience questions and discussion, breakout roundtable discussions, a panel discussion, and networking activities. We anticipate wide participation (200 attendees) by toxicologists, obstetricians, neonatologists, pediatricians, nurse specialists, endocrinologists, epidemiologists, public health and regulatory experts from basic research to clinical settings, including experts in embryonic, fetal, and childhood development, reproductive medicine and biology, and environmental toxicology. The incorporation of poster and short oral presentations by junior investigators selected from submitted abstracts, and the availability of discounted registration prices and travel fellowships will encourage participation of qualified junior investigators, fellos, physicians-in- training, graduate students, and post-docs to attend the conference. We will use targeted marketing to encourage women and minority scientists to apply for these fellowships and to participate in the conference. The discussions originating from this forum and their dissemination will foster cross-disciplinary dialogue and collaborations among complementary research and clinical groups and lead to enhanced translation of research discoveries into: (i) improved understanding of the factors involved in fetal programming in utero and pre-term birth; (ii) the identification of ways in which to predict, assess, and lower the risk of and/or prevent environmental and genetic factors that predispose to adverse fetal outcomes and pre-term birth; and (iii) related, new health and safety guidelines, and public health programs for families and children. 1 PUBLIC HEALTH RELEVANCE: Prenatal and infant environmental and pharmaceutical exposures can adversely influence fetal programming, which plays a role in pre-term birth and lasting adverse effects on pulmonary, renal, and cerebral function, behavior and learning disabilities, and vision and hearing loss and is linked to chronic adult conditions such as diabetes, high blood pressure, and cardiovascular disease. The 1.5-day scientific symposium presented by the New York Academy of Sciences and Cincinnati Children's Hospital Medical Center will provide a neutral forum for toxicologists, obstetricians, neonatologists, pediatricians nurse specialists, endocrinologists, epidemiologists, and public health and regulatory experts from basic research to clinical settings to explore recent discoveries regarding the complex array of environmental (chemical, pharmaceutical or environmental) and genetic factors responsible for fetal programming in utero and pre-term birth. This forum will generate enduring, open-access, online publications to highlight the effects of environmental exposures on embryonic, fetal, and childhood development and reproductive medicine and biology, and will be available to the global scientific community in perpetuity. Thus, this activity will foster cross-disciplinary dialogue and collaboration and promote the development of effective clinical interventions and guidelines, preventive strategies, and new policies to lower the risk of and/or prevent environmental and genetic interactions that predispose to adverse fetal outcomes and pre-term birth.

PROJECT SUMMARY (See instructions): Endometrial cancer (EMC) is the most common malignancy of the female genital tract and the fourth most common cancer among American women following breast, lung and colon cancers. Each year, about 40,000 women in the US alone become victims of EMC and about 20% of them die from the disease. The etiology of EMC is not fully understood. Several common genetic alterations are associated with human EMC. The gene with the highest frequency of alteration in EMC is Phosphatase and tensin homolog (Pten). The loss of Pten results in enhanced PISK activity and Akt activation. Increased levels of activated Akt (pAkt) stimulate both cyclooxygenase-2 (Cox2) and mammalian targets of rapamycin complex 1 (mTORCI) activity which are associated with EMC. Cox2 is over expressed in many solid tumors and Cox2-derived prostaglandins (PGs), especially PGE2 via its receptors EP2/EP4, significantly contribute to carcinogenesis. We have recently shown that levels of pAKT are elevated in Pten-deleted mouse uteri carrying EMC. Our preliminary results also show that mTORC1 activity is remarkably upregulated in mouse models of EMC. These observations suggest that Cox2-derived PGs and the mTORC1 pathway play significant roles in the development and progression of EMC and inhibiting these pathways may attenuate the incidence and/or virulence of EMC. However, the fact that long-term use of Cox2 inhibitors is associated with increased cardiovascular risks underscores the need for further investigation to circumvent those risks. There is an urgent need to build upon the current knowledge to develop new strategies in which the therapeutic index is improved. Rational combinations of low doses of these inhibitors offer the potential for improved efficacy with reduced toxicity. Our central theme is to test the hypotheses that Pten deficiency activates PI3K-pAkt-Cox2 and PI3K-pAkt-mT0RC1 pathways that together initiate and promote EMC and targeting both Cox2 and mTORC1 will be synergistic and more effective than either alone in combating EMC. We will test this hypothesis using our newly established mouse model of EMC.