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Record Count: 21
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DESCRIPTION (provided by applicant): Nuclear functions in cells depend upon critical thiols that are subject to oxidative damage by environmental, occupational and therapeutic chemicals. Thioredoxin-1 (Trx1) and glutathione (GSH) are biological thiols that protect against oxidants. These thiols also function in redox signaling and control. The purpose of this proposal is to gain an understanding of the distinct functions of Trx1 and GSH in protection against oxidative stress in cell nuclei. Aim 1 is focused on the quantitative importance of the nuclear Trx1 system compared to cytoplasmic Trx1 and GSH-dependent systems for protection against oxidative stress. Cell models will be used to examine nuclear responses to exogenous H2O2, endogenously nuclear-generated H2O2 and oxidant chemicals. Novel redox western blot and mass spectrometry proteomic techniques will be used to determine the redox state of nuclear Trx1 and S-glutathionylation of nuclear proteins. Nuclear-targeted Trx1 (NLS-Trx1) and dominant negative forms will be used to control the nuclear Trx1 system; NLS-peroxiredoxin-1 (NLS-Prx1) will be used to enhance nuclear peroxide metabolism; and cellular GSH will be modified by control of synthesis and by selective depletion. Nuclear-targeted (NLS-) D- amino acid oxidase with added D-amino acids will be used to stimulate intranuclear H2O2 generation. Toxicants will be studied to test for selective nuclear resistance/sensitivity to oxidative stress. Specific Aim 2 is to develop transgenic mouse models expressing fusion proteins with nuclear localization and nuclear export signals (NES) fused to human Trx1 (NLS-hTrx1 and NES-hTrx1) and use these for in vivo toxicologic studies of nuclear oxidative stress. Transgenic mice will be characterized for nuclear and cytoplasmic redox under control conditions and with an established in vivo toxicologic challenge with the cardiotoxicant, adriamycin. The results of these studies will provide the first test of the hypothesis that enhanced nuclear antioxidant systems protect against toxicity in vivo. Specific Aim 3 is to use mass spectrometry-based redox proteomic techniques to identify nuclear proteins controlled by the Trx1 and GSH systems and to identify nuclear proteins that are selectively vulnerable to oxidative stress. The successful completion of these aims will provide fundamental new knowledge concerning the quantitative importance of major thiol/disulfide control systems in nuclei in protection against oxidative stress from environmental and therapeutic toxicities.
DESCRIPTION (provided by applicant): Mitochondria are prone to environmental, occupational and drug-induced toxicities mediated by oxidative mechanisms. Accumulating evidence indicates that such mitochondrial toxicity contributes to neurodegenerative, cardiovascular and other chronic and age-related diseases. The present research focuses on the delineation of protective functions against such toxicities which are dependent upon the two major thiol antioxidants in mitochondria, thioredoxin-2 (Trx2) and glutathione (mtGSH). Both of these support multiple protective pathways, and both are known to protect in common toxicity models. However, it is not known whether these systems protect against distinct mechanisms in mitochondria. Our preliminary data provide evidence for dependence of toxicity mechanisms upon Trx2 under conditions where GSH was unaffected while another condition showed that GSH/GSSG was affected without a change in Trx2. These results led us to hypothesize that the Trx2 and mtGSH systems support reduction of different proteins and consequently have distinct functions in protecting mitochondria from major mechanisms of oxidative toxicity. In Aim 1, we will use mass spectrometry-based redox proteomic methods to identify physiologic mitochondrial protein substrates for Trx2 and the GSH-dependent mitochondrial glutaredoxin-2 in a doxorubicin-induced toxicity model using a cardiomyocyte cell line. In Aim 2, the role of these Trx2- and mtGSH-dependent protein systems in protecting mitochondria from toxicological insults will be tested. The goal of Aim 3 is to translate these concepts to in vivo studies of doxorubicin cardiotoxicity in mice, using models to experimentally vary the abundance of Trx2 and GSH systems. This research will advance understanding of oxidative toxicities by elucidating details of the Trx2 and GSH pathways in mitochondria and translating these to in vivo models. Given that there are limited means to protect against mitochondrial toxicities, knowledge of these pathways will provide novel therapeutic targets for interventional strategies to protect against disease processes related to chemical-induced mitochondrial dysfunction. PUBLIC HEALTH RELEVANCE: Environmental and occupational exposures can cause toxicity by enhancing the generation of reactive species and by disrupting the signaling processes required for effective regulation of energy production. While much research has focused on the reactive species, little effort has been focused on the signaling processes as a means to protect against toxicity. This is of considerable importance because therapeutic agents which eliminate reactive species could also disrupt normal signaling.
DESCRIPTION (provided by applicant)
Humans are exposed to a multitude of chemical (e.g. pollutants and drugs) and physical (e.g. radiation) agents in the environment that induce oxidative stress in most cell types that comprise tissues and organ systems. Oxidative stress, characterized by increased levels of reactive oxygen species (ROS), can induce both aberrant signal transduction and oxidative damage to cellular macromolecules, including lipids, proteins and nucleic acids. Such oxidative changes directly contribute to a variety of deleterious biological endpoints associated with several important human diseases, including cardiovascular and neurodegenerative disorders and cancer. Several lines of evidence have revealed links among oxidative stress, inflammation and the development of colon cancer. Colorectal cancer is the third most commonly diagnosed cancer and is the second leading cause of cancer death in the United States. Currently, we have a very poor understanding of the mechanisms by which oxidative stress mediates colon tumor development. The overall theme of this program project renewal is to delineate the pathogenic contributions of two major, stress-activated cellular generators of ROS (mitochondria and Nox) and to investigate the response pathways to ROS and to ROS-damaged macromolecules which directly lead to genetic instability and other biological changes that contribute to tumor development. This program of investigation builds from information obtained during the previous support period and is comprised of five complementary, synergistic projects that will employ two powerful eukaryotic model systems (yeast and mice) in order to define important elements of the eukaryotic/mammalian oxidative stress circuitry. These systems are genetically and biochemically tractable and will also provide an important mammalian intestinal tumor model that is relevant to human colon cancer development. The great advantage of employing such model systems is that key targets and system components (a number of which were identified and analyzed during the previous period of support) can be examined within the context of a battery of isogenic yeast strains, mammalian cells, and individual animals at a level of complexity and pace not yet achievable using human tissues and cells. The information generated from such studies can subsequently be directly and quickly translated into studies utilizing human material. Dissection of these cellular damages and response pathways will lead to a clearer understanding of the role of oxidative stress in colon cancer development and will reveal novel targets for prevention and intervention.
BACKGROUND
This is the first revision of a competitive renewal application for a Program Project grant that was initially funded in 2002. The overall theme of the initial proposal was to understand the interrelationships between the pathways that mediate resistance to DNA damage. The initial program consisted of five research projects. Yeast and bacterial systems were used as models. In the competitive renewal, the focus has been redirected toward colon cancer. Thus, the project that was directed by Dr. Bernard Weiss will be discontinued and replaced by a project directed by Dr. Lambeth. Based on the comments of the previous reviewers, the project by Dr. Siede has also been eliminated. Thus, the current application has four research projects. Dr. Shadel and Dr. Doetsch will direct two of the projects and the fourth project will be directed jointly by Drs. Kow and Crouse. In addition to the four research projects, there is an administrative core and a mouse tumor model and mammalian cell culture core. In the initial submission of the competitive renewal, 32 publications and manuscripts were listed as a direct result of the support provided by the program project grant. In this revised version, the number has increased to 51. The focus of the studies described in this revised application is to delineate the pathogenic contributions of the reactive oxygen species generated by the mitochondria and the Nox and the response pathways to ROS and ROS-damaged macromolecules which directly lead to genetic instability and other biological changes that contribute to tumor development. Yeast will still be used as a model system in combination with mammalian intestinal tumor models.
PROGRAM AS AN INTEGRATED EFFORT
DESCRIPTION (provided by applicant): The investigators propose to study the relationship between ambient air pollution and birth defects in the five-county metropolitan Atlanta area, 1968-2002. A growing body of evidence suggests urban air pollution may influence reproductive outcomes. In a recent study based in Los Angeles, Ritz and colleagues (2002) reported an association of certain cardiac birth defects with ambient air pollution levels estimated to have occurred during the second month of gestation, a period when the heart is developing. The proposers seek to replicate and extend this work in a new study based in Atlanta, a city with relatively high ambient pollutant levels. The study will take advantage of the availability of a rich database on ambient air quality and one of the oldest birth defects surveillance systems in the U.S. with active case ascertainment. For the 35-year study period, the proposers will obtain data on air pollution levels from the state monitoring network and several intensive air quality studies, data on birth defects from the Metropolitan Atlanta Congenital Defects Program (MACDP) operated by CDC, and data on the underlying cohort of births and fetal deaths from the Georgia Division of Public Health Vital Records. The investigators will perform temporal analyses for the period 1968-2002, and spatio-temporal analyses for the period 1980-2002, when individual-level data are available on births and fetal deaths. The primary study hypotheses relate to the specific associations reported by Ritz et al. (2002): second gestational month carbon monoxide levels and ventricular septal defects, and second month ozone levels with three other cardiac anomalies. The study will include over a million births, 38,000 birth defects, and 10,000 cardiac defects. The study will have excellent power to assess the study hypotheses, with over 99% power to observe effect sizes similar to those reported by Ritz et al. (2002), and 80% power to detect substantially smaller effects. A second major study contribution involves updating the cardiac defects classification system used by the MACDP to incorporate current embryological knowledge. The study will entail close collaboration among investigators at Emory, Georgia Tech, and CDC, capitalizing on the team's experience with the MACDP and studies of air pollution in Atlanta.
DESCRIPTION (provided by applicant): The investigators propose a targeted investigation of endocrine-related outcomes in individuals in the Michigan PBB Registry and their offspring to follow up intriguing findings of a study recently completed by this team. In 1973, inadvertent substitution of a livestock feed supplement with fire retardant led to widespread contamination of meat and dairy products in Michigan with polybrominated biphenyls (PBBs), a class of chemicals toxicologically similar to PCBs, PBDE, DDE and other halogenated organics suspected to disrupt endocrine function. Over 4000 individuals with high likelihood of exposure were subsequently enrolled into the Michigan PBB Registry, and serum samples were analyzed for PBB. The proposers recently completed a twenty-year follow up survey of women in the registry regarding occurrence of endocrine-related outcomes, and the current application will pursue key findings of this survey as follows. The observation of an association of perinatal PBB exposure with reduced age at menarche in the daughters of registry participants will be followed up in two ways: 1) an expanded assessment of ovarian function in a subgroup of the daughters, using menstrual diaries and hormonal assays of daily urine samples (50 high exposure, 50 low exposure), and 2) an investigation of pubertal development in the sons of women in the registry to assess whether analogous impacts on pubertal development are observed in the boys, using a questionnaire assessment of Tanner stages and other pubertal landmarks in sons less than 18 years of age (n=204). The suggestion of an excessive spontaneous abortion rate among the exposed daughters of reproductive age will be followed up by: 1) updating the reproductive histories of the daughters over age 18 (n=214), and 2) by determining reproductive success in the sons over 18 years of age (n=218). Finally, the finding of altered menstrual cycle characteristics among the women in the registry will, be pursued by performing a menstrual diary and urinary hormone study in the adult women (50 high exposure, 50 low exposure) similar to that proposed for the daughters. Each of the proposed investigations allows targeted, efficient follow up of the important findings observed in the previous study, and will provide further insight into possible impacts of this class of chemicals on pubertal development, reproductive health and ovarian function.
Crisp Terms/Key Words: biphenyl compound, urinalysis, human puberty, endocrine gland /system, endocrine disorder, food chain contamination, fertility, menstrual cycle, bromine, human subject, questionnaire, epidemiology, longitudinal human study, embryo /fetus toxicology, spontaneous abortion, reproduction, reproductive development, ovary, gender difference, toxicology, clinical research, personal log /diary, bioaccumulation, children
DESCRIPTION (provided by applicant): Mercury is a group IIB transition metal that is a significant environmental and occupational contaminant and hazard in the United States (and other countries). One of the primary target sites where mercuric ions accumulate and induce significant toxic effects is in the kidney. Within the kidneys, mercuric ions accumulate mainly along the three segments of the proximal tubule, although distal segments of the nephron have not been excluded as secondary targets where mercuric ions may be handled. Inorganic mercury appears to be taken up along the three segments of the proximal tubule in a heterogeneous manner. Moreover, at least two distinct sets of mechanisms are involved in the uptake of mercury along the proximal tubule. One of these sets of mechanisms is localized on the luminal membrane and the other is localized on the basolateral membrane. Based on the research we have carried out to date, we have come up with the following central hypothesis: Mercuric conjugates of biologically relevant endogenous thiol-containing molecules (or their metabolites), are 1) the primary transportable species of inorganic mercury in the kidneys, and 2) are taken up by proximal tubular epithelial cells at both the luminal and basolateral membranes by known transporters of other molecules, such as those involved in the transport of amino acids and organic anions, presumably through mechanisms involving molecular homology or "mimicry' One technology we will use to test this hypothesis will include the in vitro stable transfection of a distal-tubular, renal-epithelial, cell-line with the genetic code(s) for specific luminal and basolateral transporters presumed to be involved in the transport of mercury. Expression of a transporter in a cell that normally does contain it will permit us to determine if there is a gain of function. More specifically, we will be able to document if transport (and inhibition of transport) of specific mercuric conjugates occurs. The other technology we propose to use is the isolated perfused tubule, which is the only in vitro technique that allows one to access, perfuse, and bathe virtually any intact segment of the nephron to study the characteristics of particular transport systems under biophysical conditions similar to those found in vivo. With these techniques, we will be able to provide the most detailed and comprehensive assessment, to date, of the mechanisms involved in the uptake of inorganic mercuric ions along pars recta segments of the proximal tubule, which are the segments of the nephron most vulnerable to the nephrotoxic effects of mercury
DESCRIPTION (provided by applicant): Humans continue to be exposed to organic (CH3Hg+) and inorganic (Hg2+) forms of mercury. These environmental toxicants affect numerous organ systems, but a major target for their accumulation and toxicity is the kidney, specifically the proximal tubule. Numerous studies have shown that CH3Hg+ and Hg2+ can be removed from proximal tubules by treatment with the metal chelators, 2,3-dimercapto-1-propanesulfonic acid (DMPS) and meso-2,3-dimercaptosuccinic acid (DMSA). This removal appears to involve a conjugation step wherein intracellular mercuric ions form strong bonds with one of these chelators, and a subsequent export step where these mercury-chelator complexes are secreted into the tubular lumen. The actual mechanisms involved in this secretion, however, remain undefined. Yet based on the characteristics of the multidrug resistance proteins (MRP), it is likely that one or more of these proteins are involved in the DMPS- and DMSA- mediated export of mercuric ions. Therefore, the purpose of the current proposal is to test the hypothesis that MRP2, and/or MPR4, play a role in the DMPS- and DMSA-mediated elimination of CH3Hg+ and Hg2+ from the kidney. To test this hypothesis, we will use TR- rats, which do not express the Mrp2 protein, and Madin-Darby canine kidney (MDCK) cells stably transfected with OAT1 and MRP2 or MRP4. We will first examine the effect of DMPS and DMSA on the handling and disposition of CH3Hg+ and Hg2+ in control and TR- rats. As an alternative model, the transfected MDCK cells will be used to study the roles of MRP2 and MRP4 in the export of various species of mercury. The use of these cells allows for the separation of MRP2 and MRP4 activities and enables a more accurate characterization of the role of each protein in the transport of mercuric species. Collectively, these studies will determine whether MRP2 and/or MRP4 play(s) a role in the DMPS- and DMSA- mediated secretion of CH3Hg+ and Hg2+ from proximal tubular cells. The data obtained from these experiments will serve as the basis for a more expanded set of studies. The current and future studies will be important to human health in that they will serve as the basis for the development of additional therapeutic regimes for mercury poisoning. Mercury is a prevalent environmental toxicant to which humans are exposed frequently. The data obtained from the proposed studies will provide important information related to the way in which mercury is eliminated from the body. These data will serve as the basis for the development of additional treatments for mercury poisoning.
DESCRIPTION (provided by applicant): Organophosphates (OPs) pose a constant threat to human health due to their widespread use as pesticides and their potential employment in terrorist attacks. The acute toxicity of OPs has been extensively studied; however, the consequences of prolonged or repeated exposure to levels of OPs that produce no overt signs of acute toxicity are poorly understood. Further, there is clinical evidence that such low-level exposures to OPs leads to prolonged deficits in cognition, although the mechanism for this effect is unknown. One long- term goal of our laboratories is to elucidate the mechanisms responsible for the prolonged neurobehavioral deficits associated with chronic low-level OP exposures such that more effective therapeutic strategies can be developed. The results of our experiments conducted during the initial funding period established that low- level exposures to the commercial pesticide, chlorpyrifos, resulted in protracted deficits in prepulse inhibition (a model of pre-attentive processing) and spatial learning without significantly affecting locomotor function. Further, chlorpyrifos was associated with decreases in neurotrophin receptors and cholinergic proteins in brain regions that are important to cognitive function. These deficits were accompanied by decreases in axonal transport measured in sciatic nerves ex vivo. However, the molecular mechanisms for the deficits in axonal transport and the extent to which such effects on axonal transport occur in the brain are unclear. The objective of this application is to identify the mechanisms responsible for alterations in axonal transport as well as to further define the long-term effects of low-level OP exposure on cognitive function. Our central hypothesis is that OPs covalently modify key proteins that are involved in axonal transport and that such modifications compromise the function of neuronal pathways that support cognitive function. To achieve our objective, we propose three specific aims: 1) Determine the consequences of chronic low-level exposure to representative OPs on attention and cognitive flexibility, 2) Determine the consequences of chronic low-level exposure to representative OPs on axonal transport in the brain, and 3) Identify the molecular mechanisms responsible for OP-induced deficits in axonal transport. To address these aims, we will use a five choice serial reaction time task to assess sustained attention, a water maze task to measure extinction (a form of cognitive flexibility) and stereotaxic injections of traceable dextrans, immunohistochemistry, and mass spectrometry to determine OP effects on axonal transport in the brain and the consequences of its impairment. The significance of this project and its relevance to public health is that by mechanistically defining OPs based on their long-term effects on essential components of information processing in animals, we will have addressed a fundamental gap in our knowledge of how OPs likely affect humans over time. The experiments will contribute to a better understanding of the toxicity associated with a class of chemicals that continues to pose a significant environmental risk to millions of people worldwide. PUBLIC HEALTH RELEVANCE Organophosphates are highly toxic chemicals that are almost ubiquitous in our environment and, accordingly, they pose a significant health risk to millions of people worldwide. While the acute toxicity of these agents has been extensively studied, the effects of chronic low-level exposures to organophosphates (especially on cognition and the neuronal processes that support cognition) are poorly understood. The experiments proposed in this application have been designed to address these issues in the rodent model by mechanistically defining organophosphates based on their long-term effects on axonal transport (a fundamental process in neurons) and specific domains of cognitive function (i.e., attention and cognitive flexibility).
DESCRIPTION (provided by applicant): In an effort to delineate the mechanisms that cause the formation of inclusions and induce dopamine cell death in PD, we propose to examine a novel protein, RING-Finger Protein 11 (RNF11) and its interactions with environmental toxins associated with PD pathogenesis. Aim 1: To test that (a) RNF11 is an E3 ubiquitin ligase and (b) that RNF11 is a membrane-associated protein expressed in dopaminergic neurons. Rationale: The RING-domain is essential for recognition of E2 ubiquitin-conjugating enzyme and ubiquitination of spec- ific substrates. Expression of RNF11 in dopaminergic neurons will be suggestive of its site of E3 ubiquitin ligase activity and its role in PD. Approach: We will examine the autoubiquitination properties of RNF11, and its interaction with other proteins using GST pull down assays to determine its substrate/s in cultured cells. We will examine regional, cellular and subcellular distribution of RNF11 in normal rat and human brain by using (a) immunocytochemistry at light and electron microscope levels and (b) sub-cellular fractionation protocols to determine its site of activity. Aim 2: To test the hypothesis that RNF11 has a neuroprotective function and oxidative modification(s) of RNF11 as a result of exposure to environmental toxins will compromise its neuroprotective function. Rationale: As a component of the proteasomal degradation pathway, RNF11 will facilitate or promote clearance of proteins and increase cell viability. Exposure to environmental toxins and oxidative modifications of RNF11 could impair RNF11's role as a neuroprotectant. Approach: In cell culture systems we will examine (A) the role of RNF11 on proteasomal activity and cell survival following (i) exposure to oxidizing agents and (ii) genetic alterations including mutant RNF11 (B) Effects of structural modifications of RNF11 's subcellular distribution and E3 ligase activity and on cell viability. Aim 3: To test the hypothesis that neuroprotective function of RNF11 is compromised in PD due to depletion of functional RNF11. Rationale: Modifications of RNF11 or sequestration of RNF11 in cytoplasmic inclusions will reduce the levels of functional RNF11 and compromise its neuroprotective function. Approach: We will examine changes in RNF11 in (a) animal models of PD ie., rotenone model in rats and a-synuclein transgenic mice and (b) in autopsy brain tissue from PD patients. This study will determine a role for RNF11 and how it confers protection against dopamine cell death seen in Parkinson's disease.
DESCRIPTION (provided by applicant): Parkinson's disease (PD) is the most common neurodegenerative movement disorder characterized by progressive degeneration of dopaminergic neurons in the substantia nigra. The etiology of PD remains unknown. Epidemiological studies have revealed that exposure to environmental toxicants, including pesticides, increases the risk of PD. Many of these toxicants, such as rotenone, paraquat, and 1-methyl-4- phenyl-1,2,3,6-tetrahydropyridine (MPTP), have been shown to increase the levels of reactive oxygen species (ROS) and cause PD-like phenotypes in animals. These and other lines of evidence have implicated a crucial role for oxidative stress in PD pathogenesis. However, it is not known how environmental toxicant-induced oxidative stress leads to neuronal dysfunction and, ultimately, neuronal cell death. The long-term goal of this research is to elucidate the molecular mechanisms by which environmental toxicants cause neurodegeneration in PD. While only a very small percentage of PD cases are monogenic familial forms, molecular characterization of the identified familial PD proteins has revealed novel pathways involved in PD pathogenesis. DJ-1 is a recently identified PD gene whose mutations cause an early-onset, autosomal recessive form of familial PD. Accumulating evidence indicates that DJ-1 plays an essential role in protecting dopaminergic neurons against oxidative stress. This project will investigate the interaction between the familial PD gene DJ-1 and sporadic PD-associated environmental toxicants, and test the hypothesis that environmental toxicants cause oxidative damage to DJ-1, thereby contributing to the pathogenesis of sporadic PD in a manner similar to DJ-1 genetic mutations in causing familial PD. A combination of biochemical, proteomic, biophysical, cell biological, and molecular genetic approaches will be used to characterize the environmental toxicant-induced oxidative damage to DJ-1, examine the in vivo role of environmental toxicant-induced DJ-1 oxidation in PD pathogenesis, and determine the mechanisms by which environmental toxicants disrupt the DJ-1 neuroprotective pathway. Completion of this project should advance our understanding of the mechanistic role of environmental toxicants in neurodegeneration and help develop more effective therapies to treat PD. PUBLIC HEALTH RELEVANCE Project Narrative Although the etiology of Parkinson's disease (PD) remains unknown, there is compelling evidence that environmental toxicants, especially pesticides, are dominant risk factors in sporadic PD. The goal of the proposed research is to determine how environmental toxicants lead to neuronal dysfunction and, ultimately, neuronal cell death in PD. The results of the proposed studies will promote the discovery of new therapies for preventing and treating this devastating illness.
DESCRIPTION (provided by applicant): Parkinson's disease (PD) involves pathological loss of neurons. The long-term objective of this research in our laboratory is to understand how environmental and genetic neurotoxic agents interact to signal and regulate the survival/apoptosis machinery in PD pathogenesis. A series of our recent findings highlight the key role of nuclear cyclin dependent kinase 5 (CdkS)-mediated inhibition of survival factor myocyte enhancer factor 2 (MEF2) in neuronal survival and apoptosis. Based on this, we propose in the present application to explore the role of nuclear Cdk5-MEF2 pathway in mediating and integrating the toxic signals of PD relevant environmental toxicants and genetic risk factors in the degeneration of dopamine neurons. Our specific aims are: 1. to assess the role of nuclear Cdk5-mediatd regulation of MEF2 in environmental toxicant-induced degeneration of dopaminergic neruosn in cellular models of PD; 2. to determine the role of nuclear Cdk5- MEF2 pathway in environmemntal toxicant-induced neuronal loss in animal models of PD; and 3. to establish the role of CDk5-MEF2 pathway in integrating neurotoxic signals of environmental toxicants and alpha- synuclein in genetic models of PD. To accomplish aim Mil, we will test a group of model toxicants including MPP+(metabolite of MPTP), rotenone, and paraquat in dopaminergic neuronal cell line SN4741 cells, primary dopamine neurons, and rodents/transgenic mice to investigate whether de-regulation of nuclear Cdk5 activiy and loss of MEF2 survival promoting function underlie the demise of dopaminergic neurons in response to environmental toxicants. We will attempt to establish whether nuclear Cdk5-MEF2 mediates alpha-synculein toxicity in genetic models of PD pathogenesis and study how toxicant-alpha-synuclein interaction affects this critical signaling pathway. We will use a combination of morphological, biochemical, molecular biological, functional/behavioral and genetic methods in the proposed study. Our studies will allow us to determine whether nuclear Cdk5-MEF2 pathway is a major mediator of environmental toxicant-induced apoptosis of dopamine neurons and functions as a key converging point for the toxic effect of toxicant-alpha- synuclein interaction. This novel insight gained from this study will demonstrate how environmental toxicants and genetic risk factors may converge and disrupt a survival pathway, providing a molecular mecahnism that may underlie loss of dopamine neurons relevant to both sporadic and familial PD.
DESCRIPTION (provided by investigator): This application constitutes a renewal application for the previously funded study entitled "Analytic Methods: Environmental/Reproductive Epidemiology". The initial funding cycle facilitated a productive collaboration, and these efforts have revealed promising new directions for research to more fully encompass the multiple challenges posed by exposure and reproductive health data collected under motivating studies such as the Michigan PBB Studies (MIPBB) and the Mount Sinai Study of Women Office Workers (MSSWOW). As in many longitudinal studies, exposure assays utilized in the MIPBB underwent an evolution over time so that data obtained via the original and more recent assays are recorded at different levels of resolution. In particular, data obtained earlier in the study were primarily "heaped", due to assay limitations that effectively led values to be rounded to the nearest integer. Proper analysis of the longitudinal data should attribute the correct level of resolution to each data point, based on the assay used to record it. In epidemiologic studies, it is also common to observe highly skewed exposure data. The simultaneous features of heavy skewness, detection limit issues, changing assay resolution over time, and heaping due to rounding require flexible and innovative modeling, with the ultimate aim of improved prediction and valid determination of associations between exposure and reproductive health outcomes (Aim 1). Our research to date motivated by the MSSWOW study has identified new avenues of research into the modeling of time-to-pregnancy and menstrual cycle length data. In such studies, time-to-pregnancy is typically recorded in terms of a number of cycles as opposed to being measured in days or weeks, so that methods for discrete data survival analysis are required. Modeling innovations are needed in order to relate environmental exposures and other covariates to fertility in such contexts (Aim 2). Repeated menstrual cycle length data tend to be characterized by heterogeneity not only in average length, but in the level of variability as well. This motivates a need for flexible modeling and improved methods for classifying women into menstrual cycle length and variability subgroups, and brings attention to potential misclassification error (Aim3). This renewal application continues to seek improved analytic methods for epidemiologic research by means of an effective balance between statistical theory and application in the environmental and reproductive health areas. We consider both parametric and semi-parametric approaches, noting that both have their advantages in this context and that each approach has the potential to inform and augment the other. While intended to be of direct benefit to the motivating studies, the methods to be developed address issues that are common and fundamental enough to make them of broader interest in statistical and epidemiologic practice. PUBLIC HEALTH RELEVANCE Environmental exposures can have a major impact on various aspects of public health,
including women's reproductive health. This application aims to address multiple unique challenges in the analysis of exposure and reproductive health outcome data stemming from two landmark motivating studies. The statistical methods to be developed will also have broader implications toward public health studies that collect exposure and outcome data over time.
DESCRIPTION (provided by applicant): We propose a four-year interdisciplinary research program developing statistical methodology for disease ecology, the study of environmental and ecological impacts on disease incidence and spread. Many infectious diseases involve multiple hosts and vectors, each with unique behaviors, and each impacted by climate and landscape. The proposed research draws from the fields of ecology, conservation biology, environmental health, remote sensing, epidemiology, and global health. Geographic space links these disparate fields of inquiry and we use utilize spatial statistics to achieve our three specific aims: 1) Spatio-temporal inference for the local phylogeography of emerging diseases. We propose methods quantifying landscape impacts on the genetic structure of a virus in the ongoing disease outbreak of raccoon rabies in the eastern United States as motivation and application for methodological development, application and evaluation. Associations of interest involve geographic and genetic bottlenecks, and the impact of intervention programs in stalling disease spread. 2) Spatial inference linking disease incidence and environmental/ecological data from imperfectly measured systems. We propose to develop models linking spatial disease surveillance data to environmental landscapes, vectors, and reservoir hosts. We focus on surveillance data subject to spatially varying levels of quality, e.g., spatially varying probabilities of diagnosis and reporting. We use ongoing field data regarding Buruli ulcer in Ghana, West Africa to motivate and illustrate the proposed methodology. 3) Assess spatial design and performance criteria for the developed techniques. For the proposed techniques to have broad impact, it is essential to measure their performance in the context within which they will be applied. We focus primarily on spatial criteria: e.g., where one needs to collect more information and where methods reduce probability of detection or increase rates of false alarms. The results of this program will allow researchers to measure the impact of landscape features on the spread of an emerging disease allowing more accurate predictions of spread, planning of appropriate responses, and improved design of intervention strategies. The public health data gathered during this study will be used to design models, analytic techniques, and software that will assist ministries of health, non- government organizations, and researchers identify areas in which to focus surveillance, determine placement/enhancement of health treatment facilities, increase laboratory capacity, provide educational outreach activities, and organize trainings for healthcare workers, outreach coordinators, and laboratory personnel.SPATIAL STATISTICS FOR DISEASE ECOLOGY
PROJECT NARRATIVE
The proposed research project will develop analytic methods for mapping and quantifying the spread of infectious diseases in time and space through a complicated landscape. The project focuses on two diseases: a strain of rabies that is typically found in raccoons (but transmittable to humans) in the eastern United States and Buruli ulcer (a bacterial infection resulting in deep skin wounds) in Ghana, West Africa. The primary goal of the research is to develop accurate measurements of the impact of environmental factors (such as rivers and mountain ranges) on the spread of diseases across a diverse landscape in order to design effective, geographically specific public health responses.
DESCRIPTION (provided by applicant)
Data compiled over the last two decades indicate that chronic exposure to persistent environmental toxicants, including polychlorinated biphenyls (PCBs) and chlorinated organic insecticides, increases the risk of developing neurodegenerative disease, such as Parkinson's disease (PD). Recent studies suggest that these toxicants contribute to elevated intracellular dopamine levels, increased oxidative stress, and ultimately dopaminergic cell death, a hallmark of PD. The primary objective of this proposal is to rapidly advance the career of the candidate in this field through a combination of didactic learning, mentored research, and multi-disciplinary proposal development. The candidate, Kurt D. Pennell, is an Associate Professor of Environmental Engineering in the School of Civil & Environmental Engineering at the Georgia Institute of Technology. The career development plan, which includes coursework in bioinformatics, advanced spectroscopy and neurotoxicology, builds upon his training in science and engineering, research on contaminant transport and remediation in subsurface systems, analytical skills, and on-going sabbatical in the Department of Neurology at Emory University. The proposed activities will be carried out at Georgia Tech and Emory, where the candidate holds the title of visiting Associate Professor. The underlying hypothesis of the proposed research is that biomarkers of oxidative stress and metabolomic profiles can be correlated to chronic toxicant exposures and neurodegenerative disease. Under the mentorship of Dean P. Jones, Professor and Director of the Emory Clinical Biomarkers Laboratory, the specific aims of the research project are to: 1) Quantify the accumulation and distribution of chlorinated organic compounds in postmortem human tissue and exposed C57BL/6 mice, 2) Assess metabolic disruption and oxidative stress using established HPLC methods and H1-nuclear magnetic resonance (NMR) spectroscopy, and 3) Develop and evaluate a high-throughput, quantitative metabolomic profiling method based on coupled ion exchange liquid chromatography and Fourier Transform Ion Cyclotron Resonance Mass Spectrometry (FT-ICR MS). Dr. Pennell's career goals are to establish an active research program in environmental metabolomics, play an integral role in multi-disciplinary research teams investigating chronic exposures to environmental toxicants, and to be a leader in fostering linkages between environmental engineering and biomedical sciences.
Relevance: The proposal is relevant to public health because it will advance our understanding of the relationship between chronic exposure to environmental toxicants and neurodegenerative disease, provide quantitative methods to rapidly screen for oxidative stress and adverse metabolic profiles, and offer the possibility for early detection and intervention.
DESCRIPTION (provide by applicant)
The investigator propose a training program in environmental biostatistics addressing key and developing needs for graduate- and post-graduate trained quantitative researchers focused on the interaction between the following research themes: (a) statistical methods for environmental policy (e.g., pertaining to setting and enforcing standards for priority pollutants, quantitative risk assessment, and assessments of environmental justice concerned with differential impacts of environmental exposures across socio-demographic groups); and (b) statistical methods in quantitative disease ecology (e.g., quantifying environmental impacts on vector-borne diseases and zoonoses such as rabies and Lyme disease, including investigations of the phylo-geography or spatial patterns of particular genetic strains of such diseases). The training program integrates these two main areas through coursework and a "research rotation" for trainees. The program involves faculty from departments of Biostatistics, Environmental and Occupational Health, Epidemiology, Biology, and Law. The program includes two pre-doctoral and one postdoctoral level trainees in any given year, likely students in the Department of Biostatistics, but possibly individuals with training and interests in environmental sciences or environmental law seeking training in advanced areas of statistical analysis. The primary facilities span disciplines housed in the Graduate School of Arts and Sciences, the Woodruff Health Sciences Center, the Rollins School of Public Health, and the School of Law, all at Emory University.
DESCRIPTION (provided by applicant):
The project is to investigate radiation-induced .genomic instability, defined as a phenomenon whereby radiation related genetic damage manifests itself one or more cell generations following the generation in which damage was inflicted. Although the mechanism is not fully understood, it is believed that both DNAtargeted and non-DNA targeted effects of. radiation contribute. Genomic instability increases the risk of cancer, potentially affects tissue aging, and presents a risk to unexposed future generations. In contrast to conventional radiation effects, genomic instability has a complex and possibly nonlinear dose-response relationship, and is thus particularly relevant to low dose and low dose-rate environmental exposures. This project will identify genetic pathways that protect against radiation-induced genomic instability in vivo. It will use a novel whole-animal model, the Japanese Medaka fish (Oryzias latipes), that is similar to higher vertebrates in terms of organ systems and pattern of development and has homologs of most mammalian DNA damage surveillance and repair genes. Of relevance to work proposed here, medaka is a genetically tractable model with established technologies for transgenesis and gene silencing, where large numbers of individuals can be phenotypically screened, and with a short generation time facilitating multigenerational studies. Genomic instability will be measured in vivo based on a locus-specific test based on an unstable, engineered transgene. Individual aims are: (1) to characterize the tissue-specific dose response to low dose ionizing radiation using somatic recombination as an indicator of genomic instability, (2) to investigate how the tissue-specific response to low dose ionizing radiation is modified as a function of genetic and phenotypic background, and (3) to identify conditions under which genomic instability is transmitted to future generations and to identify factors that modify transgenerational risk. The significance of this study is to provide an opportunity to identify germline pathways of radiation response on an organism-wide scale that is not feasible in higher vertebrate models. Because of the high degree of functional gene conservation among vertebrates, mechanisms identified in the O. latipes model can readily be evaluated in mammalian species and the results then extrapolated to human. ,
DESCRIPTION (provided by applicant): Parkinson's disease (PD) is a debilitating neurodegenerative disorder, which is characterized by the loss of dopamine (DA) in the putamen and DA producing neurons of the substantia nigra, resulting in motor disturbances such as tremor, rigidity, postural instability and bradykinesia. PD is currently thought to be caused by a combination of environmental and genetic factors. Current therapies include L-DOPA administration, MAOB inhibitors, and DA agonists, which all increase the amount of DA signaling in the brain. Normally, DA is highly regulated within the neuron; the dopamine transporter (DAT) mediates selective uptake of dopamine from the synapse, adjusting the magnitude and duration of neuronal signaling. The vesicular monoamine transporter (VMAT2) then pumps cytosolic DA into vesicles for exocytotic release. VMAT2 is important in mediating neuronal susceptibility to cellular damage, particularly via the prevention of oxidative stress. VMAT2 normally sequesters catecholamines, protecting against neuronal damage; however, when VMAT2 expression is reduced, DA and NE can become oxidized in the cytosol, inducing endogenous oxidative damage. During periods of DA handling dysfunction, cellular antioxidant defenses are activated; but when these defenses fail, neurodegeneration can occur. In order to study the role of VMAT2 in PD, many groups have tried to generate VMAT2 KO mice, but completely ablating VMAT2 is lethal. Previously, our laboratory has shown that mice with a 95% genetic reduction in VMAT2 (VMAT2 LO) display progressive loss of striatal DA, L-DOPA responsive behavioral deficits, synuclein aggregation, and nigral dopamine cell loss. We hypothesize that the progressive damage in the VMAT2 deficient mice is due to increased oxidative stress as the mice age. The goal of this research is to elucidate the role of VMAT2 in the development of PD, more specifically, the ability of VMAT2 to modulate neuronal redox state in the presence of catecholamine mediated toxicity. I will test this hypothesis by 1) determining if there is an agedependent altered redox state in VMAT2 LO mice, 2) investigating if L-DOPA administration exacerbates this altered redox status, and 3) determining the role of NE in oxidant-induced neurodegeneration in the VMAT2 LO mice. Given the increasing importance of oxidative stress related research, especially as it pertains to neurodegeneration, this research has the potential to illuminate possible endogenous mechanism for the development of PD in humans. Furthermore, any oxidative mechanisms that are uncovered through this research can serve as the link between genetic and environmental factors in PD disease progression.
DESCRIPTION (provided by applicant): The World Health Organization recognizes inorganic arsenic as a carcinogen, and a serious threat to millions of people. One mechanism cells use to deal with exposure to toxicants such as arsenic is to pump them out of the cytoplasm using a special type of ABC transporter called a multidrug resistance protein (MRP). The yeast MRP, YCF1, actively pumps glutathione:arsenite conjugates into the vacuole, resulting in arsenic resistance. A YCF1 deletion strain lacks pump activity and is hypersensitive to arsenic. We propose that enhanced versions of YCF1 will confer increased arsenic uptake, accumulation, and resistance upon transgenic cells, and the Aycfl deletion strain will provide a convenient experimental system to test this hypothesis. The goal of this work is to identify conserved residues and domains within MRPs responsible for regulating arsenic toxicity, and will be addressed with the following specific aims: (1) Enhance the arsenic- transporting pump activity of YCF1. We will use PCR to randomly mutagenize the YCF1 gene, homologous recombination with a yeast expression vector to generate a library of mutants in an Aycfl yeast background, and phenotypic screening to identify mutations conferring enhanced arsenic resistance on Aycfl cells. (2) Explore the effects of corresponding mutations on human and plant YCF1 homologs. Human (MRP1, 2) and plant (AtMRPI, 3) multidrug resistance genes will be introduced into Aycfl yeast, and tested for their ability to rescue the Aycfl arsenic-sensitivity. Promising mutations identified in YCF1 will be introduced into the corresponding sites of these human and plant MRPs by site-directed mutagenesis, and the ability of these mutant MRPs to complement Aycfl yeast with enhanced arsenic pump activity will be examined. (3) Demonstrate an MRP-mediated reduction in arsenic-induced toxicity. Independent plant and CHO (Chinese hamster ovary) cell lines carrying the most promising mutant MRP versions identified will be generated and examined for acquisition of enhanced arsenic pump activity, measured by cell survival rates, arsenic accumulation, and induction of stress-related transcripts. Our coordinated work with yeast, plant, and human MRPs, in three different biochemical backgrounds, will define evolutionarily conserved regions that are important for arsenic pump activity and/or substrate specificity. RELEVANCE: This information will contribute toward dissecting the functions of the entire MRP subfamily of ABC transporters, several members of which have been implicated in human diseases, resistance to chemotherapeutic drugs, or metal toxicity. In addition, because YCF1 activity contributes not only to arsenic, but also to cadmium, mercury, and lead resistance in yeast, our results will likely be applicable toward several environmental toxicants.
DESCRIPTION (provided by applicant):
The primary purpose of this broadly based multidisciplinary toxicology training program is to prepare trainees for biomedical research careers in schools of medicine, public health, and pharmacy, and in governmental or industrial laboratories with a sound knowledge in environmental health and principles of toxicology. The areas of research emphasis represented by the 13 training faculty include neurotoxicity and neurodegenerative disease, mechanisms of cell death, oxidative DNA damage and repair, and environmental assessment and epidemiology. The 13 members of the training faculty are currently funded by over $7,000,000 in federal grants with more than a third of that being from NIEHS. Several scientists from the Centers for Disease Control, including the Agency for Toxic Substances and Diseases Registry, also participate in the training of students through guest lectures and collaborative research. The most important component of training is laboratory research, first as a series of three research rotations, then in the dissertation laboratory. This training is complemented by core courses in toxicology, biochemistry, pharmacology, cell biology, molecular biology, and biostatistics, and advanced courses in toxicology specialty areas, seminar courses, and journal clubs.
The program is designed to support four students and two postdoctoral fellows each year from a total eligible population of about 16 students and 24 postdoctoral fellows. The student who completes the predoctoral training program will have acquired broad familiarity with toxicology and environmental health, knowledge in depth in the area of dissertation research, the ability to search, read critically and report on the literature of the biomedical sciences, mastery of a variety of laboratory techniques useful in modern biomedical research, skill in planning and executing a research project, ability to write clear, accurate scientific reports for publication, and ability to present effectively the results of research. The postdoctoral fellow who completes the training program will have gained extensive experience in cutting edge toxicology research, and should have submitted an individual NRSA application to gain grant writing experience to aid in the transition into an independent research position.
DESCRIPTION (provided by applicant)
The ability to several manufactured nanomaterials to induce oxidative stress has been suggested to be the most appropriate means of assessing the potential toxicity of manufactured nanomaterials. Since oxidative stress is a common pathogenic mechanism in numerous diseases, including various neurodegenerative diseases, it is possible that the various nanomaterials may contribute to the disease process. We have shown that the redox state (dynamic balance between reduced and oxidized components) of neurons (in vitro and in vivo) can be spatially resolved by subcellular compartment. Neurotoxicants can preferentially oxidize cytoplasmic, mitochondrial, or nuclear redox components, such as thioredoxin or GSH. We hypothesize that the overall toxicity of nanomaterials will correspond to their ability to induce oxidative stress in distinct subcellular compartments and that these measures will provide a superior means of assessing their potential toxicity. We propose a series of in vitro and in vivo experiments aimed at determining the subcellular redox state of a cellular population known to be especially vulnerable to oxidative injury, namely, the dopamine neurons in the substantia nigra pars compacta. Aim 1. To determine the ability of the manufactured nanomaterials fullerene (C60), fullerol (C60(OH)22-24), manganese oxide (MnO2), titanium dioxide (TiO2), magnetic iron oxide (FeO4), and nanoscale zero valent iron (n-ZVI) to preferentially oxidize sucellular redox components. In this aim, we will examine the ability of suspended nanoparticles to induce oxidative stress in cell cultures of neuronal origin in the absence and presence of an oxidative challenge (6-OHDA). In addition, we will assess the physico-chemical properties of the nanomaterials prior to and after exposure to the cellular model. Aim 2. To determine the ability of the manufactured nanomaterials "manganese oxide (MnO2), titanium dioxide (TiO2), magnetic iron oxide (FeO4), and nanoscale zero valent iron (n-ZVI) to induce oxidative stress in dopaminergic brain regions. This aim will examine the ability of nanomaterials to alter subcellular redox state and induce oxidative damage in dopaminergic brain regions and determine the physico-chemical state of the nanomaterials prior to administration and in the brain tissue of exposed animals. Aim 3. To determine the ability of bioavailable antioxidants to attenuate the oxidative stress induced by manufactured nanomaterials. In this aim N-acetyl cysteine and alpha tocopherol will be tested for their ability to attenuate oxidative stress in in vitro and in vivo settings. Completion of these aims will provide novel information on the ability of nanomaterials to induce oxidative stress with subcellular spatial resolution and determine if their physico-chemical state is altered after exposure to the biological system.
DESCRIPTION (provided by applicant)
Parkinson's disease (PD) involves pathological loss of neurons. The long-term objective of this research is to understand how environmental and genetic neurotoxic agents interact to signal and regulate the survival/apoptosis machinery in PD pathogenesis. Mitochondrial dysfunction has been proposed as a key mechanism that mediates demise of dopaminergic neurons in PD. However, the detailed molecular mechanisms by which PD relevant environmental toxicants affect mitochondria! transcription and activity remain unknown. Recently published findings from this lab highlight the key role of nuclear transcription factor myocyte enhancer factor 2 (MEF2) in neuronal survival. Unpublished studies have revealed unexpected presence and function of MEF2 in mitochondria. Based on this, this proposal seeks to explore the role of mitochondrial MEF2 in mediating and integrating the toxic signals of PD relevant environmental toxicants in the degeneration of dopamine neurons. The specific aims are to: 1). Determine the role of mitochondrial MEF2 in regulating transcription of mitochondrial genome in dopaminergic neurons; 2). Study the regulation of mitochondrial MEF2 by PD relevant environmental toxicants in mitochondrial dysfunction and neuronal death in cellular models; and 3). Establish the regulation and function of mitochondrial MEF2 in toxicant-induced animal models of PD. To accomplish Specific Aims 1-3 the role of MEF2 in mitochondrial gene transcription will be established in dopaminergic neuronal cell line SN4741 and primary neurons and a group of model toxicants including MPP+(metabolite of MPTP) and rotenone will be tested in cellular and rodent models to investigate whether de-regulation of mitochondrial MEF2 mediates the toxic effects of these toxins. Moreover, an attempt will be to extend these findings to PD patients by correlating the levels and activity of mitochondrial MEF2 with the disease. A combination of morphological, biochemical, functional and genetic methods will be employed in the proposed study. These studies will allow an assessment of whether or not targeting mitochondrial MEF2 underlies environmental toxicant-induced apoptosis of dopamine neurons. The novel insight gained from this study will demonstrate how environmental toxicants may disrupt mitochondrial function, providing a molecular explanation for the loss of dopamine neurons that may relevant to both sporadic and familial PD and a potential therapeutic target.
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