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Record Count: 17
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DESCRIPTION (provided by applicant): Nickel-containing compounds are human carcinogens. The mechanisms of their carcinogenic actions remain to be investigated. Recent studies have indicated that reactive oxygen species (ROS) may play an important role. We hypothesize that nickel induces generation of ROS, which activate nuclear transcription factors, leading to cell transformation and tumorigenesis. Specific Aim 1 will detect and identify ROS generated in human bronchial epithelial cells (BEAS-2B) and mouse embryo fibroblast cells exposed to nickel compounds and investigate the mechanism involved. We hypothesize that nickel (Ni3S2 and NiCl2) can stimulate the cells to activate NADPH oxidase via cdc42 and p47phox to produce superoxide radical, which is then converted to hydrogen peroxide and hydroxyl radical. Specific Aim 2 will test the hypothesis that ROS are required for activation of NFAT and NFkappaB in cells and in vivo in response to nickel compounds. The role of ROS in nickel-induced activation of NFAT and NFkappaB in cells will be evaluated by co-transfection of NFkappaB-luciferase or NFAT-luciferase reporter plasmids and specific ROS scavenger enzymes. For in vivo study, BALB/c transgenic mice with alternation of antioxidant enzymes or NADPH oxidase (ROS generating enzyme) will be employed. Specific Aim 3 will investigate the role of ROS, NFAT and NFkappaB in nickel-induced cell transformation and tumorigenesis. We hypothesize that ROS activate transcription factors and cause cell transformation and tumorigenesis. We will use overexpression of DN-NFAT, DN-IkappaBalpha and DN- IKKbeta to investigate the involvement of NFAT and NFkappaB in nickel-cell transformation and induced tumorigenesis. The role of ROS will be investigated using specific antioxidant enzyme expressions and NADPH oxidase alternation. We anticipate that nickel causes activation of NFAT and NFkappaB through ROS reactions, leading to cell transformation and tumorigenesis. We attempt to link the cell transformation and tumorigenesis with specific transcription factors and specific reactive oxygen species. The results obtained from this proposal will elucidate the role of ROS and NFAT/NFkappaB signaling in Ni compounds-induced carcinogenesis. The long term goals are to provide a fundamental understanding concerning the mechanism of carcinogenic actions of Ni; to fill a need for the mechanistic information of cancer risk assessment for exposure; to propose methods for early detection; and to develop intervention and prevention strategies. Nickel-containing compounds are human carcinogens. This project will investigate the mechanism of Ni-induced carcinogenesis by testing the hypothesis that nickel induces generation of ROS, which activate nuclear transcription factors, leading to cell transformation and tumorigenesis. The long term goals are to understand the mechanism of Ni-carcinogenesis; to propose methods for early detection; and to develop intervention and prevention strategies.
DESCRIPTION (provided by applicant): Arsenic is a natural contaminant of drinking water in many parts of the world, is a known human carcinogen and is #1 on the EPA list of hazardous chemicals. Cancers most often associated with chronic arsenism are squamous and basal cell carcinomas of the skin. How arsenic causes cancer is unknown. However, the National Research Council Report on Arsenic in Drinking Water concluded that the most likely mode of action is induction of numerical and structural chromosomal abnormalities. Arsenite, the carcinogenic form of arsenic found in drinking water, disrupts mitosis causing an anaphase delay and induces aneuploidy in normal diploid human fibroblasts and peripheral blood lymphocytes, and mitotic arrest associated apoptosis (MAAA) in p53 deficient human fibroblasts. The sensitivity of p53 deficient human cells to arsenite induced MAAA suggests that the mechanism of arsenite carcinogenesis is different than sunlight induced skin carcinogenesis in which p53 mutation is an early and common event. The hypothesis to be investigated is that p53 relieves the arsenite-induced anaphase block by activation of the G2 checkpoint response which inactivates cyclin B/cdc2 and derepresses the mitotic exit network and allow the cells to escape arsenite induced MAAA. It is the prevention of apoptosis in arsenic intoxicated cells that allows genetic instability (aneuploidy) after mitotic disruption. Identification of the cellular factors that interact with p53 or the p53 regulated genes to prevent mitotic arrest associated apoptosis and to allow cells to proceed through mitosis with a delay will provide valuable information regarding the mode of action of arsenite. The specific aims proposed are: 1.) Determine activation of the G2 checkpoint pathway in p53(+) and p53(-) cells arrested by arsenite in mitosis; 2.) Test by overexpression and targeted knockdown of G2 checkpoint proteins the role of G2 checkpoint activation in the escape from arsenite induced anaphase block; 3.) Test whether arsenic associated skin tumors are p53 wild type or mutant. The results of these studies will identify players mediating release from arsenite induced mitotic arrest, and will provide valuable information on the mechanism of arsenic induced carcinogenesis, clues to the usefulness of arsenite as a chemotherapeutic agent and valuable information on the mode of action of mitosis disrupting drugs in killing human cells.
Crisp Terms/Key Words: p53 gene /protein, cyclin, apoptosis, human genetic material tag, Chinese, ultraviolet radiation, phosphorylation, antisense nucleic acid, chemical carcinogenesis, squamous cell carcinoma, basal cell carcinoma, arsenic, human tissue, aneuploidy, gene mutation, flow cytometry, cell growth regulation
DESCRIPTION (provided by applicant): The broad, long term goals of this proposal are to use a novel class of antagonists of the aryl hydrocarbon receptor (AHR), AHR-Protacs, developed herein as 1) a research tool to delineate the mechanisms by which activation of the AHR pathway by environmental agents leads to carcinogenesis in the human population and identify roles of the AHR in other disease processes and 2) a therapeutic agent to treat cancers, diabetes and cardiovascular diseases. Exposures to environmental factors, such as benzo[a]pyrene and 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD) are thought to play important roles in the development of human cancers. While these agents are known to exert many of their carcinogenic actions via activation of the AHR, the mechanisms by which the AHR elicits the key events are unknown. Further, it has not yet been established whether targeting the AHR would be an effective chemopreventive approach. Finally, emerging evidence implicates a role for the AHR in not only cancer, but also diabetes and cardiovascular diseases. The immediate goals of the current proposal are to optimize and characterize novel AHR antagonists, some of which have already been developed in our laboratories, as appropriate AHR antagonists. Specific Aims: (1) Synthesis of optimum AHR Protacs. (2) Determine that the optimum AHR Protac molecule(s) inhibits AHR signaling in cultured cells with high efficacy. (3) Determine whether the optimum AHR Protac(s) inhibits the ability of TCDD to alter cell cycle, apoptosis and senescence. (4) Determine whether the optimum AHR Protac(s) alters the AHR pathway with high specificity. The work proposed herein will develop a novel research tool that can be used to identify how inappropriate activation of the AHR by environmental carcinogens leads to human cancers and to understand the role of the AHR in normal biological processes. In addition, this work will set the stage for the development of drugs that target the AHR and may be clinically useful not only as agents that may prevent cancers, but may also be effective in the treatment of diabetes and cardiovascular diseases.
DESCRIPTION (provided by applicant): The long-term goal of this project is to elucidate the metabolic pathways that regulate the cytotoxic and mutagenic potential of base propenals (BP) generated from oxidant-induced DNA damage. BP are derived from selective hydrogen abstraction from C4' of the deoxyribose ring, and their formation leads to strand scission. High concentrations of BPs are generated by anticancer drugs such as bleomycin and environmental agents such as Cr (VI). BPs have also been detected in normal, untreated, human cells, suggesting that they are produced from background DNA damage. The BPs are highly reactive unsaturated aldehydes which readily attack cellular nucleophiles such as glutathione and guanine. At high concentrations BPs are cytotoxic and cytostatic, whereas at low concentrations they form potentially mutagenic DNA adducts. Propenal-derived DNA adducts have been detected in high abundance in normal and cancerous human tissues and it has been suggested that they are derived mainly from BPs originating from oxidant-mediated DNA damage. Nonetheless, the metabolic processes that regulate the cytotoxic and mutagenic potential of BPs are not well understood. Based on our preliminary data and literature evidence, we propose that glutathione S-transferase (GSTP1-1) and aldose reductase (AR) are the major enzymes that participate in the metabolism of base propenals and that the sequential biotransformation by these enzymes prevents the toxicity and mutagenicity of base propenals. To test this hypothesis we will identify the major cellular and urinary metabolites of base propenals generated by COS-7 and HepG2 cells or mice exposed to radiolabeled adenine and thymine propenals (Aim 1). Next we will delineate the contribution of AR and GSTP1-1 to the overall metabolism of base propenals using pharmacological and molecular strategies, and elucidate the metabolism of propenal in AR-inhibitor treated or GSTP1-1 null mice (Aim 2). To test the catalytic efficiency of AR and GSTP1-1, we will examine their steady-state kinetic properties and determine whether product or substrate inhibition limits propenal metabolism via this pathway (Aim 3). Finally, to delineate the role of GSTP1-1 and AR in preventing the acute toxicity of base propenals and their ability to form DNA adducts (Aim 4), we will test whether inhibition or overexpression of these enzymes prevents base propenal cytotoxicity and adduct formation, and whether the mutagenic burden due to base propenal exposure is altered in GSTP1-1-null or AR inhibitor-treated mice. The results of these studies will provide a better understanding of the cytotoxic effects of DNA degradation products and the mechanism by which they indirectly induce potentially mutagenic DNA lesions. Our results may also help in designing more effective and targeted anticancer interventions and could lead to the identification of organ systems and individuals more sensitive to endogenous and environmental oxidants.
DESCRIPTION (provided by applicant): The main goal of the Center for Environmental Genomics and Integrative Biology (CEGIB) is to support outstanding basic and translational investigations into the etiology of environmental disease and the development of new approaches to manage these conditions. Center investigators with diverse disciplinary backgrounds and research interests will combine their efforts to: 1) study genetic, molecular and cellular mechanisms of environmental injury, 2) evaluate the ethical, legal and social implications of genomics research, and 3) advance the emerging fields of environmental genomics and intergrative biology. CEGIB will provide scientific leadership and administrative support for creation of a synergistic inter- and trans-disciplinary network structure that brings together University of Louisville investigators to focus on the study of environmental disease. The Center will support facility cores in Bioinformatics, Biostatistics and Computational Biology, and Integrative Health Sciences. These cores will provide expertise and facilities to advance research efforts in the areas of environmental cardiology, environmental carcinogenesis, and developmental origins of health and disease. The Center structure also includes programs in Career Development for Junior Investigators and a Pilot Project Program to encourage development of novel research approaches and data collection that enable pursuit of other funding mechanisms. A Community Outreach and Education Core has been included to facilitate translation of scientific information into knowledge that can use be used to reduce health disparities of underserved Hispanic populations in the greater Louisville area. CEGIB is in a unique position to meet its objectives given the depth of expertise and talent in environmental health, molecular biology, bioinformatics and computational biology and ELSI research, and strong institutional emphasis on bench-to-bedside and bench-to-community investigations. The focus on environmental genomics and integrative biology reflects the interests of its members in advancing molecular investigations and ELSI research of environmentally related diseases. The creation of a Center with a resolute focus on environmental genomics and integrative biology will further NIEHS' goal to understand the etiology of environmental disease at its most fundamental levels and provide a visible infrastructure for advancement of the Institute's agenda in the post-genomic era.
DESCRIPTION (provided by applicant)
The University of Kentucky is unique among land grant universities in that all colleges, including Medicine and Agriculture, are located on the same campus. This constellation of programs has enabled the UK-SBRP to develop uniquely productive collaborations across diverse disciplines. Such an environment will allow to study the overall theme of the SBRP research, which focuses on the toxicology of Superfund chemicals and how health effects of exposure can be modulated by both intrinsic and extrinsic factors, namely genetics and nutrition, respectively. Given the abundance of Superfund chemicals and widespread distribution in the ecosystem, it is unlikely that remediation alone will be sufficient to address their health risks. Nutritional intervention thus becomes a sensible way to address health problems associated with environmental pollutants. In the competing renewal, the investigators recognize this dual need for sensing/remediation and biomedical intervention through nutrition by proposing five integrated projects. The investigators will concentrate on chlorinated organics (e.g., polychlorinated biphenyls) prevalent in most Superfund sites, including those found in Kentucky. Preliminary findings by this group suggest that nutrition and dietary habits can markedly influence mechanisms of toxicity of the above-mentioned Superfund chemicals. Thus, a major objective of our SBRP is to explore the paradigm that nutrition can modify Superfund chemical toxicity. All biomedical projects will focus on chronic diseases associated with vascular dysfunction, such as cardiovascular disease, cancer metastasis, and obesity-related abdominal aortic aneurysms, and will utilize a similar dietary fat regimen to study nutrient/toxicant interactions. There will be significant cross-talk with non-biomedical projects, which will explore novel techniques for both remediation (detoxification) and biosensors associated with detection of PCBs and other chlorinated organics. Results from interdisciplinary research will be utilized for information/education, technology transfer, training, policy and translational purposes as part of the objectives of the Research Translation, Community Outreach, and Training Cores. Nutrition may be the most sensible means to develop primary prevention strategies of diseases associated with many environmental toxic insults. Thus, research proposed by the investigators may lead to novel dietary recommendations at the national level for populations at risk, thus improving the health of people residing near Superfund sites.
ADMINISTRATIVE CORE
Description (provided by applicant)
The Administrative Core (Core A) will oversee the entire Superfund Basic Research Program through a management mechanism that allows efficient and accountable oversight and maximum interdisciplinary and multidisciplinary interactions among all participants. The Administrative Core will be responsible for the coordination, planning, assessment, project accounting, and administrative support to the research personnel of the University of Kentucky SBRP. A major objective of the Administrative Core is to facilitate the execution of an interdisciplinary research theme through administrative support and fiscal accountability. The Administrative Core will work especially closely with the Research Translation Core (Core C) to optimize communication with NIEHS, EPA, and state and local constituents; communication is also facilitated by an active website that is managed by Core C with input from the Administrative Core. Research efforts will be coordinated through biweekly meetings of faculty, students and research staff, as well as seminars, where new findings and planned activities and ideas will be discussed. It will also be critical to discuss research findings for informative/educational activities, technology transfer, training, policy and translational purposes as part of the objectives of the Research Translation, Community Outreach, and Training Cores. The Administrative Core will implement feedback and cross-talk between all project and core activities to allow for maximum integration. For example, optimal detection and remediation of Superfund pollutants must be linked to risk assessment and biomedical aspects of degradation products. Accountability of the University of Kentucky SBRP activities, including the quality of science and administration, will be achieved through regular consultations with both the External and Internal Advisory Boards. Research quality assessment and statistical consultation will be managed through the Research Support Core. General fiscal responsibilities or project accounting will be coordinated through the Administrative Office in conjunction with the University of Kentucky Research Foundation. The Program Coordinator together with the Program Director will serve as a regular contact among all SBRP activities, including research projects and cores, and will coordinate all reports required by the sponsoring agencies.
This Program-Project Grant is to identify and define the cardiovascular effects of environmental aldehydes and pollutants that generate aldehydes. The project will integrate molecular and cellular aspects of aldehyde toxicity, delineate the contribution of individual pathways involved in the detoxification of the aldehydes, and elucidate how aldehydes affect atherosclerosis, platelet and endothelial activation, and myocardial function. Acrotein and trans-2-hexanal will be studied
as model aldehydes most prevalent in the environment. The Program consists of 4 Projects and 3 Cores. Project 1: Aldehyde Metabolism and Cardiovascular Disease focuses on the metabolism of these toxicants in mice. These studies are designed to identify the major metabolic products and the biochemical pathways that metabolize aldehydes, and how glutathione S-transferases regulate this metabolism and cardiovascular toxicity. Project 2: Atherogenic Effects of Environmental Aldehydes will test the hypothesis that exposure to unsaturated aldehydes exacerbates atherogenesis. Spontaneously atherosclerotic apoE-null mice will be exposed to these pollutants, and changes in lesion progression and markers of vascular inflammation will be measured, and resultant alterations in plasma lipoproteins will be determined. The central hypothesis of Project 3: Role of Platelet Activation and Vascular Pathology in Aldehyde Toxicity is that environmental aldehydes activate blood cells and induce proinflammatory responses in the vascular endothelium. To test this hypothesis, we will examine platelet activation by aldehyde, investigate the role of platelet microparticle formation in aldehyde-induced vascular changes, and assess the ability of these aldehydes to stimulate platelet-lesion interactions. In Project 4: Acute and Chronic Cardiotoxicity of Aldehydes will examine how aldehydes acutely depress contractibility, exacerbate ischemic injury, and chronically induce inflammatory states contributing to remodeling characteristic of heart failure. For this, we will delineate the mechanisms by which aldehyde inflict contractile dysfunction, abolish ischemic preconditioning and induce pathological myocardial remodeling. The Administrative Core (A) will serve as the organizational focus and will provide infrastructure support, including statistical analysis, data
management, coordinated sample handling and storage and sentinel monitoring for systemic toxicity. The Bioanalysis Core (B) will provide and integrated, centralized facility for biochemical determinations and mass spectrometry. The Inhalation Facility Core (C) will provide uniform acrolein exposures to mice via inhalation. Successful completion of this project will lead to a better understanding of the cardiovascular consequences of aldehyde exposure and will provide direction to future assessments of human risk and susceptibility.
DESCRIPTION (provided by applicant): Cardiovascular disease is the major cause of death in the U.S. Chronic arsenic ingestion causes atherosclerosis and is associated with increased mortality from myocardial infarction and stroke. Early life arsenic exposure may play a significant role in development of atherosclerosis in adult life, as suggested by reports of infant deaths from myocardial infarction caused by advanced arteriosclerosis in regions where mothers consumed water with high levels of arsenic. Transplacental arsenic exposure can disrupt liver DNA methylation patterns and thus alter liver gene expression. Underlying liver disease is an independent risk factor for atherosclerosis. Hence, arsenic exposure induced liver disease may predispose to atherogenesis, and widespread exposure to arsenic in drinking water in the U.S. likely contributes to atherogenesis and death from cardiovascular disease. Our working hypothesis is that arsenic exposure disrupts epigenetic control of hepatic gene expression predisposing the liver to inflammation that is an atherogenic trigger resulting in accelerated atherosclerosis in susceptible animals. Preliminary data indicate that atherosclerosis-prone apolipoprotein E knockout (ApoE -/-) mice transplacentally exposed to arsenic in drinking water develop overt vascular disease by 10 weeks of age without high lipid diet and that liver gene expression suggests a pro-inflammatory state. The aims of this exploratory grant application are 1) to refine and to optimize this newly developed model of transplacental arsenic induced atherogenesis by determining arsenic exposure response of arsenic blood levels in pregnant females and fetuses, determining exposure/time dependence of atherosclerotic lesion formation (rate and extent of lesion formation), assessing arsenic exposure-response of changes in vascular reactivity and characterizing the nature of arterial lesions (lesion cellularity, fibrosis and inflammation) in arsenic exposed mice; 2) explore transplacental arsenic-induced hepatic changes by characterizing liver pathology, determining circulating biomarkers of liver dysfunction and test the hypothesis that there is a temporal correlation between hepatic changes and vascular changes. Tissues will be examined at birth and at 10, 16 and 24 weeks of age to determine the course of disease and stability of molecular changes induced by in utero arsenic exposure. The goal of the proposed studies is to determine whether there is a threshold level of arsenic exposure necessary to accelerate atherogenesis using the ApoE model. The relevance of this model to human atherosclerosis is that people prenatally exposed to arsenic are likely more susceptible to dietary influences later in life. These studies will provide important information on the mechanisms of arsenic induced atherosclerosis and the role that fetal arsenic exposure plays in disease progression. This model also will be a rich resource for future research on the mechanism of arsenic induced atherosclerosis including determination of the critical stages of development for the arsenic effect, and molecular studies of arsenic exposure induced changes in DNA methylation, chromatin structure, gene expression and genetic imprinting. PUBLIC HEALTH RELEVANCE: Exposure to arsenic drinking water is a major concern in the United States. The effect of early life arsenic exposure on adult disease progression is unknown but potentially very important. This project uses a mouse model of fetal arsenic exposure induced vascular disease to investigate effects of fetal arsenic exposure on liver development that likely cause vascular disease by young adulthood.
DESCRIPTION (provided by applicant): The long-term goal of this research is to investigate the fetal origins of breast cancer and the role of environmental factors during early development, conception-weaning. 2 sources of estrogenic exposure relevant to man will be studied: a phytoestrogen preparation from soy mainly containing genistein and daidzein, and the ubiquitous environmental xenoestrogen 4-nonylphenol. Our hypothesis states the maternal estrogenic environment has a strong influence on risk to mammary tumorigenesis in adult life. 2 corollaries are: (a) phytoestrogens (90 ppm genistein/daidzein), in conjunction with an isocaloric 20% fat-diet having high n-3/n-6 polyunsaturated fatty acid balance, will significantly lower the risk of mammary tumors; and (b) xenoestrogen exposure (25 ppm 4-nonylphenol), in conjunction with a low dietary n-3/n-6 fatty acid balance, will promote the risk. The hypothesis will be tested in a unique mouse model for breast cancer having mammary-specific expression of a conditional mutation in the tumor suppressor Trp53 allele (flox'd p53.R270H/WAP-Cre model). The "humanized" mutant allele is permanently activated in the mammary gland epithelium exclusively in females traversing their first pregnancy-lactation cycle. This expression predisposes females to mammary tumors (-70% incidence) between ages 25-52 weeks. The project has 2 concurrent Specific Aims. (1) To investigate the effects of gestational-neonatal exposure to phyto/xenoestrogens on mammary gland development and tumorigenesis in female offspring heterozygous for the conditional p53.R270H allele reared on diets of low and high n-3/n-6 fatty acid composition. (2) Using the powerful combination of gene expression profiling with laser capture microdissection enumerate the molecular abundance profiles of epithelial end-buds and subtending ducts as foci for pre-malignant changes in the naive (virgin) and initiated (parous) mammary gland of p53.R270H/WAP-Cre females. These studies will provide new biomarker leads based in genetic regulatory networks that correlate (or anti-correlate) with altered fetal programming and pre-malignancy together with the realization of how such networks might relate functionally to the prenatal environmental risk factors in breast cancer.
Crisp Terms/Key Words: laboratory mouse, genetically modified animal, polymerase chain reaction, gas chromatography mass spectrometry, hormone regulation /control mechanism, estrogen, molecular oncology, breast neoplasm, cancer risk, tumor promoter, dietary lipid, mother /embryo /fetus nutrition, nutrition related tag, longitudinal animal study, embryo /fetus, placental transfer, early experience, mammary epithelium, animal genetic material tag, phytoestrogen, microarray technology, laser capture microdissection, developmental disease /disorder, bioinformatics, gene expression profiling
DESCRIPTION (provided by applicant): Environmental exposure to UV light is a major etiologic factor in the development of human skin cancers. Experimentally, UV acts both as tumor initiator and a tumor promoter in animal models. It is recognized that certain nutritional factors and phytochemicals play an important role in an individual's susceptibility to environmental carcinogens. Understanding of the underlying mechanisms will enhance the effectiveness of prevention and therapeutic efforts. Our preliminary studies indicate that apple peel extract has antioxidant activities. It inhibits ultraviolet (UVB, 280-320 nm) radiation-induced AP-1 activation both in vitro and in vivo, possibly by interfering with signal transduction events involving MAP kinase, ERKs, and JNK. Cell transformation studies show that apple peel extract inhibited TPA-induced cell transformation. We hypothesize that apple peel extract may function as an antioxidant and as a chemopreventive agent against oxidative stress-induced carcinogenesis. We will use UVB-induced skin carcinoma as a model to test this hypothesis. Four specific aims are proposed. Specific Aim 1 will identify the active antioxidant components of apple peel extract. We will study the antioxidant properties of various chromatographic fractions derived from apple peel extract and examine the phenolic compounds identified previously for their reaction rates toward oxygen free radicals. Specific Aim 2 will investigate antioxidant properties of apple peel extract, individual fraction, and previously identified phenolic compounds in JB6 cells and in living animals. Specific Aim 3 will investigate the effects of apple peel extract on UVB-induced activation of AP-1, NF-DB, and NAFT in vitro and in vivo. Specific Aim 4 will evaluate the effects of apple peel extract, individual fraction, and previously identified phenolic compounds on UVB-induced lipid peroxidation, protein oxidation, oxidative DMA damage, and carcinogenesis in SKH-1 hairless mice exposed to UVB. This Aim will also investigate the effects of apple peel preparations on proliferative and/or cell deletion activities by measuring proliferation and apoptosis. This application can be used as a preclinical study for nutritional intervention against cancer resulting from environmental exposure to UV light. This study is related to the priority areas of the Public Health Service program, "Healthy People 2010," which is committed to achieving the promotion of health and prevention of disease. Thus, the results may have an important impact on public health.
DESCRIPTION (provided by applicant): Lung cancer, the leading cause of cancer-related deaths in America, is most commonly caused by long-term exposure to tobacco smoke carcinogens such as benzo[a]pyrene (BaP), which is metabolized into the ultimate metabolite benoz[a]pyrene-7,8-dihyrodiol-9,10-epoxide (BPDE). Despite improvements in the treatment and management of lung cancer, these methods fail to reduce the overall mortality rate of this disease. While dietary components such as curcumin (turmic spice) show promising effects against BPDEinduced carinogenesis, its exact mode of action is not fully understood. The objective of this project is to elucidate whether pretreatment with curcumin inhibits BPDE-induced DMA damage by activating p53. We hypothesize that curcumin may reduce the mutagenic activity of low dose BPDE by lowering the threshold of p53 activation, thereby inducing DNA repair and cell cycle arrest at lower BPDE exposures. In Specific aim 1 the expression of p53 and p53-regulated proteins as well as cell cycle progression will be monitored by Western blot and flow cytometric analysis, respectively, to determine whether curcumin influences cell cycle arrest in response to BPDE exposure. Because global genomic repair (GGR) removes most BPDE-DNA
adducts in a p53-dependent manner, Specific aim 2 will use immunoslot blot, [32P]-postlabeling, and
UvrABC mediated gene and strand-specific southern blot assays to analyze whether curcumin affects p53 activation and thus the relative extent of BPDE-DNA adducts formed and removed in genomic DNA. In Specific aim 3 Northern and Western blot assays will be used to investigate the role of curcumin in the induction of damage recognition proteins of the GGR pathway (XPC, DDB2), which are p53-dependent. Furthermore it is believed that conjugation of glutathione (GSH) to BPDE by glutathione-S-transferase (GST) interacts with the expression of p53 to inactivate BPDE. In Specific aim 4, Western blot and GST colorimetric assays will be used to monitor the expression of proteins involved in the GSH detoxification system as well as the overall activity of GST, respectively. This aim will provide a better understanding of potential alternative routes that may inactivate BPDE and how curcumin may influence this activity. By investigating the effects of curcumin on p53-mediated responses to DNA damage, a better understanding of the potential role of curcumin in preventing tobacco carcinogen-induced mutagenesis and carcinogenesis will be gained. In addition, the results from this project will benefit the public by providing easy and inexpensive alternative methods in preventing initial and continuing signs of lung cancer.
DESCRIPTION (provided by applicant)
The primary objective of the Summer Education Experience in Research (SEER) in Toxicology is to provide 6 students with an opportunity to participate actively in ongoing research together with an educational seminar series that provides a background in research in environmental health sciences. The immediate goals are to provide a research experience for students at a critical time of career decision-making and to develop the research skills and expose the students to the potential and excitement of research careers. The long-term goal is to recruit these students into the Toxicology doctoral program. Students will have hands-on responsibilities in research projects in which reasonable conclusions can be obtained in a 9-week period, and will spend 85% of their time in the laboratory. Research opportunities in specialty areas of Oxidative Stress, DNA Repair and Mutagenesis, Biochemical Toxicology, and Chlorinated Organics and Cardiovascular Toxicity are provided by a group of 17 faculty members with experience in mentoring graduate and undergraduate students. Students will work with faculty who are primarily Core and Joint Faculty in the Graduate Center for Toxicology and who are Training Grant Faculty on the Molecular Mechanisms of Toxicity NIEHS T32 Training Grant or the Superfund Training Core. Students will be recruited from the University of Puerto Rico (UPR) as the continuation of a program for students from UPR that has been cosponsored by the University of Kentucky (UK) and UPR for about 9 years, and was supported by an NIEHS T35 program from 2001 - 2006. The investigators will work with experienced faculty from the UPR to identify and recruit students for the SEER Toxicology program. Applicants must provide their student transcripts and will be interviewed by the UPR faculty to identify those with the maturity and academic excellence to succeed in a summer research experience. The UPR faculty will send the applications of the best students to Dr. Vore, who will review them along with 2 other UK faculty mentors, and select the top 6 applicants. This group will also match the interests of the applicants with those of the faculty who are able to work with the students in any given summer. A seminar series that exposes students to the broader issues in the environmental health sciences is also provided, along with seminars on the responsible conduct of research, and a presentation of how to prepare and apply for Graduate School. Students will make a 15-minute presentation of their research findings in a symposium-type format at the end of the summer. A plan for evaluating student satisfaction, the effectiveness of mentors and the program has been developed and will be used to enhance the program in ensuing years.
DESCRIPTION (provided by applicant): This project will determine whether the mechanism by which arsenate induces mitotic effects on the Anaphase Promoting Complex (APC) mediate arrest. We hypothesize that arsenite inhibits the APC and subsequently mitotic progression. The APC orchestrates progress through the cell cycle and mitosis by targeting specific mitotic substrates for proteasomal degradation via ubiquitination. Specific aim 1 will determine whether arsenite treatment inhibits APC substrate degradation necessary for mitotic exit. In Specific aim 2 we will immunoprecipitate (IP) the APC and identify its subunits in order to investigate arsenite binding to APC subunits using radiolabeled Na[73As]O2. Determination of arsenite binding requires resolution of APC subunits using SDS-PAGE, examination of protein banding pattern using Sipro-Red and fluorescence imaging, and subsequent autoradiography to elucidate bands incorporating radiolabeled arsenite. Western blot analysis of SDS-PAGE resolved APC subunits will be used to identify specific APC subunits. Once arsenite association is determined, the proteins will be excised from the gel and identity confirmed by mass spectrometry. Specific aim 3 will determine whether arsenite inhibits APC ubiquitin ligase activity. Isolated APC will be used in an ubiquitin conjugating assay with radiolabeled APC substrates treated with or without arsenite. Reaction products will be resolved on SDS-PAGE and autoradiography will be used to determine the degree of substrate laddering due to ubiquitin conjugation. Identification of the molecular targets of arsenite is important for chemotherapeutic drug development.
DESCRIPTION (provided by applicant):
This proposal describes an environmental health sciences predoctoral training program at the
University of Louisville. It is a dynamic partnership between the Departments of Biochemistry &
Molecular Biology and Pharmacology & Toxicology, both of which have long-standing successful graduate programs. The training program benefits from their combined faculty, research, and programmatic strengths. The state-of-the art training program is enhanced through active participation and interaction with six interdisciplinary research centers: 1) Center for Environmental and Occupational Health Sciences 2) Center for Genetics and Molecular Medicine, and 3) Institute for Public Health Research, 4) Birth Defects Center, 5) James Graham Brown Cancer Center, and 6) Institute for Bioethics, Health Policy and Law. Trainees rotate through faculty laboratories to receive interdisciplinary research training and to select dissertation research projects focusing in four areas of environmental health science research: 1) environmental genomics, 2) basic mechanisms of environmental insult, 3) reproductive health and 4) environmental cardiology. A core curriculum provides an interdisciplinary academic foundation in biochemistry and molecular biology, pharmacology and toxicology, cell biology, genetics, epidemiology, biostatistics, research methods and bioethics. Initially student recruitment and funding for predoctoral trainees is provided by the University of Louisville School of Medicine Integrated Programs in the Biomedical Sciences recruitment gateway. Funds are requested to support two predoctoral trainees/year beginning in their second year. The training program will begin with four trainees and increase to eight trainees as the program matures. Several interdisciplinary program projects and core facilities also strengthen student training. The University of Louisville has increased its commitment to health science research and infrastructure, graduate education, and minority recruitment, making this training program particularly timely.
DESCRIPTION (provided by applicant)
The goal of the Molecular Mechanisms of Toxicity Training Program is to provide students with an education in toxicology that is based on an understanding of biochemistry, physiology, pharmacology and molecular/cell biology, coupled with an in-depth research experience on the mechanisms by which specific agents induce toxicity, and/or the basic cellular processes upon which environmental agents impact to cause toxicity. The Training Program is based in the Graduate Center for Toxicology (GCT) which grants the Ph.D. in Toxicology, and provides an administrative and teaching nucleus of 6 core faculty. The diversity of training opportunities is enhanced by 24 joint faculty who have primary appointments in the Colleges of Medicine, Pharmacy, Agriculture and Arts & Sciences, and Nutritional Sciences. Numerous collaborations and multidisciplinary approaches to research problems among this group provide a rich research training environment. This application requests support for 8 predoctoral trainees in five areas of strength which serve as a focus: 1) Oxidative Stress, 2) DNA Repair, 3) Biochemical Toxicology, 4) Neurotoxicology and 5) Immunotoxicology. Current enrollment in the Toxicology Program is 35 predoctoral students, including 5 minority students. The University of Kentucky sponsors a program with the University of Puerto Rico to bring up to 10 students for summer research experiences as a means of increasing our recruitment of outstanding minority doctoral students; 6-7 of these students work in the laboratories of GCT faculty and 5 are supported by a T35 Environmental Toxicology/Short Term Research Grant (ES 10951). The predoctoral training program requires 23 credit hours in biomedical based coursework (i.e., biochemistry, physiology, cell biology, pharmacology, statistics), 13 in toxicology-based courses (including Ethics in Scientific Research) and two electives (3-4 credits) selected from courses germane to the research area (e.g., molecular biology, neuroscience, immunology). Students are guided through the program initially by a Director of Graduate Studies, followed by a Major Advisor and a 4-member Advisory Committee, which supervises the student's Qualifying Examination, research and thesis defense. There continues to be strong University support for the GCT in the form of student fellowships, supplementation of the Training Grant, and new faculty lines, space and equipment. The program continues to grow in terms of the quality and breadth of the Training Program due to recruitment of new faculty with vigorous research programs, increased funding of faculty and increased enrollment of excellent, motivated predoctoral students.
DESCRIPTION (provided by applicant)
This proposal describes a short-term, summer training program for medical students focused on exposing students to career opportunities in basic and clinical research related to environmental health sciences. This application addresses 2 specific aims: Aim 1: To provide hands-on training in basic and clinical environmental health sciences-based research to medical students in a structured mentored environment. Aim 2: To provide an interactive, educational experience that introduces medical students the fundamental skills necessary for basic, translational, and clinical environmental health sciences-based research. The training program will include didactic instruction as well as a hands-on-research experience under the direction of a team of experienced basic and clinical research scientists centered on a core of NIEHS-funded investigators and collaborating faculty. These faculty members have long-standing success in mentoring medical and graduate students and post-doctoral fellows. Students who have completed at least one year of medical school will be eligible to apply for this 10-week training program. Trainees will be selected based upon their academic performance, letters of recommendation, and matching their research interests and career goals with suitable mentors. The investigators have been successful in recruiting students from minority, rural, and economically disadvantaged backgrounds. The students will participate in an established 10-week didactic summer program: "From Bench to Bedside: Introduction to Clinical Research", which included bioethics, as well as Environmental Health Sciences-specific seminars and journal clubs in basic and clinical research. Students will present posters of their research at the end of the program. The training program will begin with six trainees and increase to ten trainees as the program matures. Over the past 6 years, the University of Louisville has increased its commitment to health science research and infrastructure, graduate education, and minority recruitment, making this short-term training program for medical students particularly timely.
DESCRIPTION (provided by applicant): Acute high-level exposures to chemicals that damage the respiratory tract can cause life-threatening lung injury. Chlorine gas is a highly toxic respiratory irritant that when inhaled causes cellular injury, alveolar- capillary barrier disruption, inflammation, and pulmonary edema. We are investigating mechanisms by which G proteins, which are ubiquitous intracellular signaling molecules, regulate lung injury, inflammation, and repair. During the course of the proposed research we will investigate how G protein-mediated signaling pathways regulate injury and inflammation that are induced when lungs, or lung cells, are exposed to chlorine gas. We will then apply the information gained from these studies to develop novel treatment strategies based on modulation of G protein function to ameliorate acute lung injury. G protein coupled receptors (GPCRs), which control cellular homeostasis and responses to environmental stimuli, are activated by a variety of neuropeptides, inflammatory mediators, and hormones that are released following tissue injury. GPCRs activate intracellular signaling pathways by stimulating G proteins that have been classified into four families: Gq, Gs, Gj, and Gi2. We have observed that activation of Gq in lung epithelial cells stimulates proinflammatory gene expression, whereas activation of Gs promotes increased survival following injury. In the proposed experiments, we will use manipulation of Gq and Gs signaling pathways as potential therapeutic measures to treat acute lung injury induced by inhalation of chlorine gas. In Specific Aim 1 we will examine mechanisms by which Gq signaling promotes activation of the proinflammatory transcription factor NF-KB and Gs inhibits chlorine toxicity in cultured epithelial and endothelial cells. In Specific Aim 2, we will determine, using an inducible, cell-specific knockout mouse model, whether Gq signaling in lung epithelial cells is a therapeutic target for ameliorating acute lung injury. In Specific Aim 3, we will develop treatment strategies, including cell-soluble Gq inhibitory peptides and Gq siRNA, for chlorine-induced lung injury based on inhibition of Gq function. In Specific Aim 4, we will optimize the in vivo delivery of therapeutic agents for chlorine-induced lung injury based on inhibition of Gq function and stimulation signaling pathways downstream of Gs. This application is submitted in response to RFA-NS-06-004, "Countermeasures Against Chemical Threats." The proposed experiments are designed to understand how inhalation of a toxic chemical injures the lung and, based on this information, to develop novel ways to treat or prevent acute lung injury. This type of research is sought through the RFA because of concerns that U. S. civilians could be adversely affected by highly toxic chemicals released intentionally in terrorist attacks or unintentionally in industrial accidents or natural disasters.
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