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Record Count: 6
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DESCRIPTION (provided by applicant): The aryl hydrocarbon receptor (AhR) is an orphan nuclear receptor and a central mediator of the effects of an entire class of environmental toxicants. The AhR has an undefined role in normal and disrupted embryogenesis as well. Reproductive senescence is accelerated by 2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD, dioxin), the specific and potent AhR ligand, at environmentally relevant exposures and one consequence is a decline in oocyte and subsequent embryo quality. As women increasingly delay attempts at pregnancy into their 30s and beyond, any acceleration of reproductive aging has dire consequences for the possibility of successful pregnancy and the risk of birth defects. Furthermore, early reproductive senescence in women is associated with decreased lifespan and quality of life. Although the effects of AhR ligands on oocyte numbers have been studied extensively, little is known about the AhR, oocyte quality and subsequent organization of the early embryo. In our preliminary work, we have identified early chromosomal and cytoskeletal defects that may be pathognomonic for exposure of oocytes and embryos to AhR ligands such as dioxins; this can explain the decreased fertility resulting from chronic exposure with age. The objective of this project is to identify checkpoints of oocyte development and early embryogenesis that are regulated by AhR ligands. Our central hypothesis is that environmental exposure to AhR ligands reinforces the decline in oocyte and embryo quality with age. This work, once completed, promises new insights to prevent the loss of fertility in polluted environments and slow the loss of fertility with age. The research team will achieve these experimental goals through the following specific aims: Specific Aim 1: Determine the effect of defined AhR activation on critical periods of oocyte maturation and early embryogenesis in vitro. This aim encompasses the most detailed investigation into the impact of AhR activation or antagonism on the loss of oocyte and embryo quality to date. Endpoints include embryonic morphology, cytoskeleton and chromatin and its epigenetic modulation in oocytes and embryo. Specific Aim 2: Determine the impact of chronic, environmentally relevant AhR activation and antagonism on oocyte and embryo quality during normal and accelerated reproductive senescence. Oocytes and embryos from young and middle aged rats will be assessed for morphological and cytoskeletal conformation and chromatin remodeling and epigenetic modification and imprinting from oogenesis to early embryogenesis. This will provide insight into decreased oocyte and embryo quality and fertility with age and the importance of the AhR pathway in the aging ovary and oocyte. This work is novel through use of realistic exposures to AhR agonists and a focus on subtle measures oocyte and embryo quality and epigenetics. These studies explore a novel diagnostic indicator of compromised oocyte and embryo quality due to exposure to toxic AhR ligands. This project will provide crucial knowledge for the identification and prevention of infertility and birth defects due to the AhR pathway in older mothers. Hundreds of manmade compounds including dioxins, polychlorinated biphenyls and carcinogens in cigarette smoke act on cells through the aryl hydrocarbon receptor (AhR) pathway. We have recently reported detrimental effects of AhR activation on aging of the ovary and a number of defects in eggs and embryos from females exposed to AhR ligands. This project will provide crucial knowledge for the identification and prevention of infertility and birth defects due to the AhR pathway in older mothers.
DESCRIPTION (provided by applicant): Elucidating the mechanisms by which chemicals and drugs are taken up into and eliminated from cells can enhance the understanding of drug bioavailability and can aid in prediction of tissue-specific distribution and toxicity of drugs. Organic anion transporting polypeptide (Oatp) and multidrug resistance-associated protein (Mrp) transporters mediate uptake and efflux, respectively, of a wide variety of substrates in liver. Coordinate regulation of uptake and efflux transporters may be a mechanism by which cells protect themselves from chemicals, for example, by simultaneously decreasing entrance and enhancing elimination of chemicals. Perflourodecanoic acid (PFDA) is a ten-carbon fluorinated fatty acid and a component of numerous commercial products. PFDA is a peroxisome proliferator that also produces a broad spectrum of toxicity in rodents. Our own experiments have demonstrated that PFDA causes unique and dramatic changes in hepatic expression of Oatp and Mrp transporters. These PFDA-mediated changes in transporter expression are likely to have physiological implications by affecting hepatic uptake and elimination of both endogenous and xenobiotic substrates. These novel effects of PFDA on transporter gene expression thus merit further investigation. Therefore, PFDA can be used as a powerful tool to elucidate mechanisms of transporter regulation. To further our knowledge of transporter regulation by PFDA, we propose the following studies: 1) examine alterations in transporter gene expression as a function of both dose and time, 2) determine the effects of these changes in gene expression on xenobiotic distribution, and 3) examine the contribution of several key transcription factors in the regulation of transporters by PFDA. The studies proposed will increase our understanding of the unique and dramatic effects of PFDA on transporter genes and on the consequences of altering transporter gene expression. More generally, these investigations will elucidate mechanisms of transporter gene regulation, information that is important in predicting possible therapeutic benefits of transporter modulation, as well as possible drug-drug interactions in humans.
DESCRIPTION (provided by applicant): Multidrug resistance-associated proteins (Mrps) play a key role in hepatic detoxication by transporting Phase-ll conjugates and other organic compounds out of hepatocytes. Similarly, upregulation of Mrps in tumor cells confers resistance to chemotherapeutic drugs by transporting these cytotoxic compounds out of cells. The regulatory mechanisms governing Mrp expression in normal and diseased liver, and in tumor cells, are not understood. Thus, the overall goal of this application is to study the molecular mechanisms of transcriptional regulation of Mrps. Nuclear factor E2 related-factor 2 (Nrf2) is emerging as a critical transcription factor in regulation of both constitutive and inducible expression of Phase-ll enzymes. Because Mrps play a key role in the efflux of Phase-ll conjugates, we hypothesize that Mrps are coordinately regulated with Phase-ll enzymes by Nrf2. We recently examined Mrp expression in three different models: 1) mice treated with monofunctional inducers that selectively upregulate Phase-ll enzymes, 2) bile-duct ligation, a surgical model of extrahepatic cholestasis, and 3) mice with targeted disruption of glutathione synthesis. In each model, induction of Mrps and classical Nrf2 target genes was observed. Moreover, using in-silico analysis, we identified putative Nrf2-responsive sequences, known as electrophile response elements (EpREs), in the 5' flanking regions of the Mrp2, 3, and 4 genes. We hypothesize that activation and subsequent binding of Nrf2 to these EpREs results in increased expression of Mrp2, 3, and 4. Thus we propose Mrps belong to the battery of Nrf2-regulated detoxication genes. To test this hypothesis, we will determine: 1) the role of Nrf2 in Mrp induction in mice, taking advantage of Nrf2-null mice, 2) Nrf2 activation and subsequent translocation to the nucleus, 3) critical response elements by in vitro and in vivo reporter gene assay in combination with promoter deletion analysis, 4) Nrf2 binding to EpREs identified in Mrp 5' flanking regions, and 5) specificity of Nrf2 binding to Mrp promoter regions. Data from the experiments in this proposal will provide novel insight into the transcriptional regulation of Mrps. Elucidation of the role of Nrf2 in the regulation of the efflux transport process will have significant ramifications in toxicology, xenobiotics disposition, drug-drug interaction, and cancer chemoprevention.
DESCRIPTION (provided by applicant): Cellular uptake is a fundamental phenomenon required for endogenous compounds and xenobiotics, such as drugs and environmental pollutants, to elicit physiological, pharmacological, and toxicological events within the cell. One might expect that the mechanism(s) by which xenobiotics are transported into hepatic parenchymal cells will affect hepatocellular biotransformation and biliary excretion. There are numerous transport mechanisms putatively responsible for hepatic sinusoidal uptake of organic molecules. These mechanisms include sodium-independent transport of a broad range of organic anions mediated by organic anion transporting polypeptides (Oatps). The Oatp sinusoidal transporters constitute an important organic anion transport system that we postulate will be regulated by classical enzyme inducing chemicals and bile acids. Despite much progress made in cloning and identifying Oatps, there is only a limited understanding of the regulation and function of Oatps. This deficiency, coupled with the emergence of the mouse genome sequence and the availability of numerous knockout mouse models, provides us an unprecedented opportunity to study and understand the regulation of Oatp gene expression. In parallel, our newly developed Oatp4-null mouse (the first and only Oatp-null mouse) gives our laboratory a unique tool to examine functions of this important Oatp in vivo. Therefore, the current proposal represents our plans to: (1) determine the molecular regulatory mechanisms responsible for both constitutive and altered expression of the Oatp gene family in liver, and (2) characterize the in vivo function of the liver-specific transporter Oatp4. The data generated regarding the expression and regulation of Oatps, as well as functional data from our Oatp4-null mouse will greatly advance our knowledge concerning the importance of Oatps in physiology, pharmacology, and toxicology, and ultimately not only aid the scientific community in predicting drug efficacy and safety in humans, but allow the development of liver-specific drug delivery.
Crisp Terms/Key Words: nuclear receptor, detoxification, toxin metabolism, drug receptor, membrane transport protein, biotransformation, liver metabolism, liver cell, gene expression, enzyme substrate, enzyme induction /repression, drug interaction, drug metabolism, cholanate compound, biological transport, genetically modified animal, laboratory mouse
DESCRIPTION (provide by applicant)
During the past 25 years, over 200 students have received instruction in the Toxicology Training Program at the University of Kansas Medical Center. Among the trainees were 13 who received Master's degrees, 96 who received Ph.D. degrees, and 126 postdoctoral fellows. Contained in this proposal is a request for five additional years of funding for this program. In support of this, details are provided on recruiting and retaining students, including minority fellows. Also included are changes in the Program over the past decade, including modifications in and modernization of the curriculum. Plans for the next five years include increased efforts to recruit qualified students and fellows, a change in the administrative structure and oversight of the Program, a decrease in the amount of required coursework, the recruitment of new Program faculty, and the relocation of the Program to a new state-of-the-art research building. While faculty and course requirements continue to change to meet the needs of trainees, the Program emphasis will remain on familiarizing students with environmental issues, risk assessment strategies, and modern laboratory techniques of relevance to toxicology. Special emphasis is placed on teaching communication skills necessary for effective oral presentations and for preparing research reports and proposals. All doctoral students and postdoctoral fellows receive didactic and laboratory instruction in both basic and industrial research. The past decade has witnessed changes in the Program to modernize the training, to improve recruitment, to streamline and update courses, to increase the number of participating faculty, and to enhance student exposure to outside leaders and opportunities in the field. With the anticipated opening of the new research building in Summer 2006, and the recruitment of 10 additional faculty (six were recruited the last two years) in the Department of Pharmacology, Toxicology and Therapeutics, the Program is well positioned to enhance its international reputation as a center of excellence for training in toxicology.
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