Peter Thomas, Ph.D. - US grants

The objective of the proposed research is to determine whether a teleost fish, Atlantic croaker, is a reliable model for investigating the effects of chemicals on reproductive endocrine function in female vertebrates. This will be achieved by comparing the effects of lead, benzo(a)pyren, Aroclor 1254 and clomiphene (chemicals representing four classes of reproductive toxins) on reproductive endocrine function in croaker in previously published data on their effects in mammals. The synthesis and actions of gonadal steroids as well as their conjugation and excretion will be investigated both in vivo and in vitro using modern endocrinological techniques. These techniques include radioimmunoassay, radio receptor assay, in vitro steroid biosynthesis from radioactive precursors and separation of products by HPLC and TLC, in vitro oocyte maturation bioassay. Effects of chemicals on the actions of gonadotropin will also be determined using two in vitro bioassays. Indices of reproductive impairment such as ovarian growth, oocyte maturation and ultimate hatching success will also be assessed in order to determine the signficiance of these pertubations in endocrine fucntion. The accumulation of the model compounds by ovarian tissues and oocytes will be measured to determine whether a correlation exists betweent he chemical burden in the organ and the degree of reproductive endocrine dysfunction. These studies will provide valuable new information on the sites and mechanisms of toxicant action on reproductive endocrine function in vertebrates. A long-term goal of this research is to develop this teleost model as an alternative method for assessig the effects of chemicals on endocrine and reproductive function in vertebrates. Potential advantages of using this model are (a) lower costs for animal care, (b) greater public acceptance as test organisms, (c) large amounts of ovarian tissue for in vitro testing, (d) applicability as an early-warning indicator of pollution damage to aquatic ecosystems and the potential reproductive hazards of environmental contamination to human populations.

The overall objective of the proposed research is to determine the primary sites and mechanisms by which several different classes of reproductive toxins impair endocrine function in female vertebrates. The principal aim of this research is to identify common mechanisms of reproductive endocrine toxicity in vertebrates which will be of predictive value for future clinical and epidemiological studies on reproductive hazards of chemicals to humans. The actions of cadmium, lead, a polychlorinated biphenyl mixture (Aroclor 1254), an antiestrogen (clomiphene) and an estrogenic pesticide (chlordecone) will be investigated in a comprehensive teleost model of female reproductive endocrine function, the Atlantic croaker (Micropogonia undulatus). The following possible mechanisms of endocrine toxicity will be examined in detail: interference by heavy metals with second messenger systems; alterations of neuroendocrine function; enhanced metabolic clearance of steroid hormones; and interference with the molecular actions of steroid hormones. In particular, it is proposed to test the hypotheses that (1) cadmium and other heavy metals interfere with the calcium-dependent phosphoinositol system for gonadotropin secretion and the adenylate cyclase system for gonadal steroid secretion; (2) lead and Aroclor 1254 act primarily at the hypothalamus to disrupt reproduction. Moreover, it is proposed to compare the effects of Arochlor 1254 treatment on the production and metabolic clearance of steroids in vivo in order to determine the physiological significance of enhanced steroid metabolism. Finally, the mechanisms by which antiestrogens interfere with the molecular actions of estrogen will be examined in an in vitro assay of estrogen gene expression. The effects of the model compounds on reproductive endocrine function in croaker will be compared to previously published data on the effects of these chemicals in mammals to further examine the utility of this alternative animal model for predicting long-term reproductive hazards of chemical to humans. Potential advantages of using this model include: (1) greater predictive value for reproductive hazard assessment when used in combination with mammalian studies; (2) greater public acceptance as test organisms; (3) lower costs for animal care; (4) more precise investigations of gametogenesis and the molecular actions of steroid hormones; (5) applicability as an early-warning indicator of pollution damage to aquatic ecosystems and the potential reproductive hazards of environmental contamination to human populations.

9322284 WHITLEDGE This project is to develop a REU program, specifically targeted towards the participation of minority and women undergraduate students. Special emphasis will be placed on recruiting students from the HMCU's from the South Texas region. There is a wide range of projects with research opportunities and there is a good plan to integrate students with the general student population at UTMSI by the assignment of a graduate student mentor.

DESCRIPTION (provided by applicant): The long-term goal of the proposed research is to determine the principal causes of neuroendocrine disruption and targets of the major environmental contaminants of public health concern. In this continuation proposal the proximal cause of Aroclor 1254 (PCB)-induced impairment of tryptophan hydroxylase (TPH) activity and the reproductive neuroendocrine consequences will be investigated in a fish model, Atlantic croaker. The following overall hypothesis will be tested in croaker: PCB-induced impairment of the serotonin-gonadotropin releasing hormone-luteinizing hormone (5-HT-GnRH-LH) neuroendocrine pathway controlling reproduction involves a non-coplanar congener-induced decrease in TPH activity, which is the result of oxidative effects/damage. Initially, the effects of a range of PCB concentrations on TPH activity and hypothalamic levels of the TPH protein and its mRNA will be investigated followed by experiments to investigate associations between oxidative damage and neuroendocrine disruption. Subsequently, the effects of vitamin E (an antioxidant) treatment on serotonergic functions, and its efficacy in reversing the effects of the PCB on lipid peroxidation, malondialdehyde-protein adduct formation, TPH activity, and neuroendocrine function, will be investigated. Finally, the effects of a coplanar dioxin-like PCB congener (PCB 77) on TPH activity will be compared to those observed with two non-coplanar di-ortho-substituted PCB congeners (PCB 47 and PCB 153) to identify likely causative agents of the neurotoxic effects in the PCB mixture. The consequences of PCB congener-induced impairment of hypothalamic serotonergic function on the functional integrity of the 5-HT-GnRH-LH neuroendocrine system will also be investigated. The specific objectives are to: i) determine whether the PCB-induced decrease in the hypothalamic TPH activity is accompanied by alterations in TPH protein and mRNA levels; ii) determine whether the PCB-induced decreases in TPH activity and 5-HT-GnRH-LH function are associated with oxidative damage, and the efficacy of an antioxidant in reversing these effects; iii) determine whether the non-coplanar di-ortho-substituted PCB congener component of PCB mixtures could account for their toxic effects on TPH activity and 5-HT-GnRH-LH function. There is now compelling evidence that the environmental or occupational exposure to PCBs or consumption of PCB-contaminated fish in the Great Lakes is associated with reproductive and neuroendocrine dysfunction in humans and developmental deficits in their children. However, the mechanisms of PCB neurotoxicity remain poorly understood. The proposed study will investigate a novel mechanism of PCB neurotoxicity and neuroendocrine toxicity which may also have significance for other neural functions such as those associated with mental health.

Steroid hormones elicit responses in many target tissues by binding to specific receptors on the cell surface. However, lack of information on the structures of these steroid membrane receptors and their genes has prevented significant progress in our understanding of this mechanism of steroid hormone action. Recently a novel gene has been identified in spotted seatrout ovaries which appears to have the characteristics of an estrogen membrane receptor. The goal of this project is to complete the characterization of this putative estrogen membrane receptor, including its binding to steroids, localization in estrogen target tissues, activation of cellular responses, and regulation. This is a high risk project because it will not be known if the novel seatrout gene is a functional estrogen membrane receptor until all the research objectives have been completed. It is probable that a knowledge of the structure of the seatrout estrogen membrane receptor can be used to identify steroid membrane receptors in other species, including humans. Therefore the identification of the first steroid membrane receptor gene and knowledge of its structure is likely to have a major impact in many areas of biology and medicine including endocrinology, psychiatry, immunology, cancer, and reproductive disorders.

DESCRIPTION (provided by applicant): Long term goals are to determine the mechanisms of nonclassical steroid actions mediated by a novel family of putative membrane progesterone (P4) receptors (mPRs) we recently discovered in human cells and their functional significance in health and disease. Nonclassical P4 signaling pathways mediated via mPRa and mPRb in human myocytes will be investigated. Preliminary studies suggest these mPRs are upregulated in human myocytes during labor and activate multiple inhibitory G-proteins and second messenger pathways, resulting in down-regulation of nuclear progesterone receptor (nPR) transcriptional activity as well other conditions favoring myometrial contraction at term. Therefore, the hypothesis that P4 acts via the novel putative mPRa and mPR(3 receptors in human myocytes to activate multiple inhibitory G-protein/second messenger pathways and to modulate nPR transcriptional activity will be tested. Aims are to: (1) Determine steroid binding characteristics and localization of human wild type and recombinant mPRa and mPRb. Binding of progestins to cell membranes from human myocytes in the presence or absence of siRNA for the mPRs, and to mammalian cells transfected with human mPRs will be examined;(2) Investigate coupling of the mPRs to G-proteins and their associated second messenger pathways. Coupling of G-proteins, their identities and identities of second messengers activated will be investigated in these mPR cell expression models and in myometrial biopsy tissues (3) Investigate functional domains of hmPRa for steroid binding and G-protein coupling. Receptor domains of mPRa required for ligand binding and G-protein coupling will be investigated by developing pharmacophore and receptor models, followed by site-directed mutagenesis and functional analyses. (4) Explore modulation of nPR transcriptional activity and co-activator functions via mPR-dependent pathways. Cross-talk will be investigated using several reporter assays and nPR phosphorylation and co-activator function by immunological methods. Preterm birth is a major medical problem, occurring with 12% of births, but the mechanism of a functional progestin withdrawal in humans resulting in the onset of labor is unclear, The present study will characterize previously unrecognized multiple signaling cascades initiated by progesterone activation of mPRs that are likely involved in functional progesterone withdrawal at term, shifting the balance from a quiescent state to a contractile one. Although many of the specific experiments were difficult to follow, it appears that the overall organization attacks the issue on a broad front, such that a lot of information about the receptors will be forthcoming. It could be argued that this is just the type of characterization that has been done with many receptors. However, it is important to establish these points for the mPR. In addition, the studies should provide indications of important regulatory events that might occur in human cells.

There is growing concern over the recent dramatic increase in the incidence of seasonal hypoxia (low oxygen levels) in coastal regions worldwide due to increased nitrogen inputs through human activities. The impact of this environmental deterioration on the abundance of marine animals and valuable coastal fishery resources is unknown because basic information is lacking on the long term reproductive effects of hypoxia. Preliminary results suggest the presence of a specific mechanism in the brain of an estuarine fish, Atlantic croaker, to shut down reproduction in response to hypoxia by decreasing hormone secretion. The hypothesis that hypoxia impairs reproduction in croaker through down regulation of tryptophan hydroxylase (TPH), a critical enzyme for synthesis of serotonin, a chemical in the brain that regulates reproductive hormones, will be investigated using a variety of biochemical and molecular techniques. The results are expected to show that hypoxia impairs reproduction by a specific mechanism involving down regulation TPH activity in the brain. Moreover, it is predicted that the decrease in TPH activity is due to a decline in TPH mRNA expression which is a specific response to hypoxia and is not observed with other environmental stressors. This research is likely to have broad impacts on our understanding of reproductive adaptations of organisms to environmental changes, particularly to oxygen-limited conditions such as hypoxia, at high altitudes, and during hibernation, and for the many other physiological functions of serotonin. The demonstration that the down regulation of reproductive and endocrine functions by hypoxia is mediated through a hitherto unrecognized specific adaptation mechanism would suggest a major influence of hypoxia in the evolution and ecology of estuarine organisms such as croaker. However, the results of this study may have the greatest impact in formulating policy decisions, because identifying the precise mechanism by which hypoxia causes reproductive suppression in fish provides a compelling argument for the existence of this hypoxia effect and its ecological importance. Two graduate students will be supported off this project and receive training in all aspects of the research. In addition, a minority undergraduate student will be recruited for a summer research internship to conduct a senior project on hypoxia effects in croaker.

Recently there has been a dramatic increase in the number and size of low oxygen (hypoxia) regions in coastal waters worldwide due to human activities, but the long-term impacts of this global change on coastal fisheries and ecosystems are unknown. Marked reductions in egg production and hormone levels are found in Atlantic croaker collected from hypoxic coastal regions which are associated with inhibition of the brain hormone controlling reproduction, gonadotropin releasing hormone (GnRH). These studies suggest hypoxia may target three enzymes in the brain critical for GnRH function, tryptophan hydroxylase, aromatase and neuronal nitric oxide synthase. This project will test the hypothesis that all three enzymes in croaker brains are altered by hypoxia exposure through a specific mechanism resulting in down-regulation of GnRH and reproductive functions. The results are expected to show, for the first time in any vertebrate, the existence of a specific brain mechanism by which hypoxia prevents reproduction as an adaptive mechanism to conserve energy. The results will be valuable for making policy, management and regulatory decisions by resource protection agencies. Undergraduate, graduate and postdoctoral researchers, including underrepresented Hispanic minorities, will conduct independent projects and receive training on all aspects of the research and its analysis, the results of which will be communicated via different media to the general public, high school students, teachers and scientific community. Trainees will be taught how their basic research findings can be utilized to benefit society by developing their skills in recognizing the potential broader societal impacts of their research.