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Record Count: 23
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DESCRIPTION (provided by applicant): We hypothesize that a group of oxidative DNA damages, including 8-oxoguanine, formamidopyrimidines and tandem lesions, play important roles in the etiology of human cancer via the mutagenic and genotoxic effects of these lesions in certain important sequences of cancer genes. In this application, we propose to study the biological effects of a subset of these lesions. We have three specific aims. In aim 1, synthesis and characterization of oligonucleotides containing tandem base lesions in DNA will be carried out. Tandem lesions formed by ionizing radiation, transition metal/H2O2, or other radical- mediated processes include abasic site adjacent to 8-oxoguanine, S- and R-8,5'-cyclo-2'- deoxyguanosine, and intrastrand vicinal guanine-thymine cross-link. We shall determine the structural effects of these damages in DNA by thermal melting, circular dichroism, and NMR. In aim 2, single stranded plasmid or viral vectors containing these lesions will be constructed. Mutagenicity and repair studies of the site-specific lesions will be carried out in Escherichia coli and in mammalian cells. For example, we shall determine how mutagenicity of 8-oxoguanine might be influenced when it is present as part of a tandem DNA damage. We shall investigate if Fapy.dG, the guanine-thymine vicinal cross- link, S- and R-8, 5'-cyclo-2'-deoxyguanosine are mutagenic, genotoxic, or both in mammalian cells. In aim 3, we shall construct duplex plasmid vectors with these lesions, which will be used to examine the extent of lesion bypass by extracts of normal and repair- or replication-altered mammalian cells (e.g., isogenic mouse embryonic fibroblasts and cells that express modulated levels of DNA polymerases encoded by REV1, REV3, RAD18, etc. genes). We hope to contribute toward a deeper insight of the effects of oxidative DNA damages.
DESCRIPTION (provided by applicant): Bloom's syndrome (BS), an autosomal recessive disorder, is characterized by short stature, sunlight sensitivity, an increased susceptibility to infections and respiratory illness, and a strong disposition to a wide spectrum of cancers. The mean age of death is 27 years and the cause is most frequently cancer-related. At the cellular level, BS exhibits highly elevated levels of sister chromatid exchanges, interhomologue recombination, and ectopic recombination, indicative of homologous recombination (HR) deregulation. It has been proposed that abnormal HR events lead to the general destabilization of the genome in BS, manifested as chromosome aberrations and rearrangements including breaks, quadriradials, and translocations. BLM, the protein encoded by the gene mutated in BS, is a member of the RecQ helicase family. BLM has the ability to unwind DNA substrates including those that resemble HR intermediates, and it works in conjunction with Topo III alpha, a type IA topoisomerase, to dissolve the double Holliday junction (DHJ) to yield non-crossover recombinants. BLM also associates with a novel polypeptide BLAP75 and binds the hRad51 recombinase. Importantly, our preliminary studies have found a dramatic stimulatory effect of BLAP75 on the reactions mediated by BLM-Topo III alpha. This research project comprising molecular studies under three specific aims strives to decipher the biological significance of the BLM-Topo III alpha-BLAP75 (BTB) complex with regard to the processing of HR intermediates, defining HR pathway choice, and regulating the activity of hRad51. Specifically, we will (1) delineate the functional roles of BLM, Topo III alpha, and BLAP75 in the BTB complex needed for HR modulation, (2) define the contributions of BLAP75-mediated ligand interactions to BTB complex functions, and (3) test hypotheses concerning the functional significance of the BLM-hRad51 interaction. The results from our research endeavors will shed mechanistic light on BLM-dependent control mechanisms of HR and will provide an explanation for the genome instability and cancer susceptibility in BS patients.
DESCRIPTION (provided by applicant): Environmental allergenic disease is a major cause of illness and disability in the US, and there is broad consensus that the prevalence of type I allergy is increasing worldwide. In spite of the substantial societal and health costs, the methods used to detect and quantify allergens in environmental health studies limit measurement to a few well characterized allergens that represent less than 10% of total allergen diversity. Recent advances in biotechnology have yielded potentially useful functional binding biomolecules (aptamers) that can enable low cost, high affinity allergen measurement. Aptamers are selected in vitro from combinatorial oligonucleotide libraries and therefore have several advantages over the traditionally used antibodies for detection of allergens. Among these advantages are higher stability, binding affinities greater than or equal to monoclonal antibodies, a dramatic decrease in production and assay cost, and the ability to target specific IgE binding epitopes of an allergenic protein. The overall goal of this research is to determine the feasibility of using aptamer-based methods for measuring environmental allergens. Two core development issues must be addressed to achieve this goal, and they form the basis for the first two specific aims. First, aptamers that are specific for the whole allergenic protein and aptamers that are specific for an IgE epitope binding peptide within the allergen will be produced via the SELEX method and their binding affinities characterized. Alternaria mold (Alt a 1) and dust mite (Der p 1) allergens will be targeted. Next, the aptamers will be tested in an inhibition type assay to determine cross reactivity, limits of detection, and the ability to mimic IgE epitope binding. Integrating the resulting aptamer-based allergen measurements to enhance quantitation in an ongoing and complementary environmental childhood asthma epidemiological study forms the basis for the third and final aim. Correlations between allergen specific IgE blood levels and environmental level of these allergens measured by antibody-based, aptamer-based, and specific IgE-based methods will be produced. Successful use of aptamers for measuring environmental allergens should lead to a more cost effective, flexible, and health relevant method and thereby provides the potential for a more fundamental understanding of the role of environmental allergens in respiratory health.
Allergenic disease is a major cause of illness and disability in the US. In spite of the substantial health and monetary costs, the methods used to study allergens in the environment are underdeveloped. The purpose of this research is to test the feasibility of a more flexible and low cost allergen measurement method (aptamers). The successful application of this aptamer technique should lead to a more cost effective and health relevant allergen detection method and enable a better understanding of disease caused by and exacerbated by environmental allergens.
DESCRIPTION (provided by applicant): Genetic instability is a hallmark of cancer. We have discovered that the hypoxic tumor microenvironment is an important cause of this instability. We have determined that hypoxic stress causes decreased expression at the transcriptional level of the DNA mismatch repair (MMR) factors, MLH1 and MSH2, and of the homology- dependent repair (HDR) factors, RAD51 and BRCA1. The MMR and HDR pathways are critical for maintaining genomic integrity. They play important roles in repairing environmental and endogenous DNA damage, and they influence the response of tumor cells to many cancer therapies, including ionizing radiation and alkylating and cross-linking agents. Mutations in MLH1 and MSH2 have been linked to hereditary colon cancer, while mutations in BRCA1 are associated with hereditary breast and ovarian cancers. In addition, MLH1 and BRCA1 levels are reduced in many sporadic cancers of these and other sites, a process associated with epigenetic silencing. Mechanistically, our initial studies have linked the Myc/Max network of transcription factors to the regulation of MLH1 and MSH2 expression and the E2F family of factors to the regulation of BRCA1 and RAD51. In this competing renewal application, we propose hypothesis-driven, mechanistic studies to elucidate the stress- response pathways that modulate the expression of these MMR and HDR repair genes. Using a battery of genetic and biochemical techniques, our work will focus on transcriptional regulation, epigenetic chromatin modification, and signal transduction events that occur in response to hypoxia and that may regulate DNA repair. We will also test specific hypotheses regarding the influence of hypoxia-induced changes in DNA repair on DNA damage signaling and on the efficacy of tailored therapeutic strategies that exploit these DNA repair changes. The altered regulation of DNA repair in response to cellular stresses such as hypoxia may be a key cause of genetic instability that promotes carcinogenesis and fosters tumor progression. At the same time, it may also offer the possibility of devising therapeutic strategies to which hypoxic cancer cells, but not normoxic, non- malignant cells, are especially susceptible. Hence, the proposed work will contribute to our understanding of basic cancer biology and may provide the basis for new approaches to cancer therapy.
DESCRIPTION (provided by applicant): Mutations in the BRCA2 gene cause familial breast and ovarian cancers of a high penetrance, engender a strong risk to pancreatic and other cancers, and can also lead to the cancer-prone syndrome Fanconi anemia. BRCA2-deficient cells exhibit elevated chromosomal aberrations and are hypersensitive to ionizing radiation, DNA alkylating agents, and DNA crosslinking agents. Genetic studies have shown an involvement of BRCA2 in homologous recombination (HR) and the HR-mediated repair of damaged chromosomes. BRCA2 binds ssDNA and associates with the Rad51 recombinase, which catalyzes DNA joint formation in HR reactions via a filamentous polymer assembled on ssDNA. Several lines of evidence have implicated BRCA2 in the assembly of the Rad51-ssDNA nucleoprotein filament, and our preliminary studies revealing a recombination mediator activity in a BRCA2-derived polypeptide have provided compelling experimental support for this premise. To dissect the hierarchy of protein-protein and protein-ligand interactions that underlie the HR function of BRCA2, the following molecular studies will be carried out. Specifically, we will (1) define the DNA binding properties of BRCA2, (2) ascertain the functional significance of the BRCA2 DNA binding activity and of Rad51-BRCA2 interaction by characterizing variants that harbor mutations in the DNA binding domain and determining the functionality of the 8 BRC repeats of BRCA2, and (3) test hypotheses concerning influence of the BRCA2-associated protein DSS1 on the DNA binding and recombination mediator activities of BRCA2 and RPA displacement from ssDNA. Our research endeavors will help delineate the HR and tumor suppressor functions of BRCA2 and provide a paradigm for understanding the action mechanism of other recombination mediators. The experimental systems devised herein are highly suited to assessing the functional consequences of BRCA2 mutations in cancer susceptible individuals. Our studies are therefore highly germane for cancer etiology, as the molecular information derived from them can be exploited to advance cancer diagnosis, prognosis and treatment.
Crisp Terms/Key Words: nuclear protein, protein protein interaction, gene targeting, brca gene, DNA binding protein, nucleic acid repetitive sequence, DNA repair, genetic recombination, gene mutation, molecular genetics
DESCRIPTION (provided by applicant): Exposure of cells to ionizing radiation and other carcinogenic agents induces DNA double-strand breaks (DSBs). Genetic studies in Saccharomyces cerevisiae and other organisms have revealed that homologous recombination (HR) and non-homologous DNA end joining (NHEJ) represent the major mechanisms for DSB elimination. During recombinational repair, ssDNA tails derived from break processing are bound by HR factors. The nucleoprotein complex thus formed conducts a search to locate an undamaged DNA homolog and catalyzes the formation of heteroduplex DNA joints with the homolog. The RAD50, RAD51, RAD52, RAD55, RAD57, RAD59, MRE11, and XRS2 genes are key members of the evolutionarily conserved RAD52 epistasis group that mediate mitotic and meiotic recombination and the recombinational repair of DSBs. The proteins encoded by MRE11, RAD50, and XRS2 are associated in a complex that plays roles in both HR and NHEJ. Rad51 protein, with the aid of ancillary factors, nucleates onto the ssDNA tails to form a nucleoprotein filament that has the ability to initiate heteroduplex DNA formation.
In our renewal project, a combination of biochemical and genetic approaches will be used to (i) delineate the functions of MRE11/Rad50/Xrs2 in DSB repair, (ii) continue dissecting the mechanism of action of the Rad51-associated complex in heteroduplex DNA formation and chromatin remodeling, and (iii) define the multifaceted role of the Srs2 helicase in HR and DSB repair. Our studies will be important for deciphering the mechanism and functional significance of DSB repair in eukaryotes. Since defective DSB repair causes several human cancer-prone syndromes, the results from our project will be highly germane for understanding the biology and etiology of radiation-induced and chemical-induced carcinogenesis.
DESCRIPTION (provided by applicant): Heavy metal cations such as cadmium are widely found in the environment and can act as potent immunosuppressive agents. One of the biological responses to many immunosuppressive toxicants is the production of stress response proteins. Metallothionein (MT) is an intriguing example of this group of proteins, and plays several critical roles in cellular homeostasis. MT acts as a reservoir of essential metals, as a potent anti-oxidant, as a protein that can sequester toxic heavy metals and as a regulator of several transcription factors. These functions implicate MT in metal-mediated immunomodulation. We have shown that MT can significantly influence immune functions in vivo and in vitro. The fundamental premise of this proposal is that a functional immune response exists in the context of an optimum level of MT. When MT levels are elevated beyond this optimal range by toxicant exposure, we predict that significant declines in immune function will occur. We plan to evaluate these hypotheses by using two recently derived mouse strains that are both congenic with C57BL/6J. The transgenic MT strain has multiple Mt1 genes that drive MT overexpression, and the second carries targeted disruptions of the Mt1 and Mt2 genes. Our specific aims are:(1) to test the hypothesis that manipulations of metallothionein gene dose will alter the immunosuppressive consequences of exposure to cadmium, (2) to test the hypothesis that metallothionein overproduction will decrease the available oxidant in leukocytes and diminish oxidant-related damage to leukocyte plasma membranes in cells harvested from animals exposed to immunosuppressive doses of cadmium, (3) to test the hypothesis that toxicant-induced metallothionein will alter the sub-cellular distribution and tissue distribution of essential and toxic metals to immune organs and cells in the cadmium-exposed animal, and (4) to test the hypothesis that metallothionein gene dose in an animal will influence the signal transduction cascade and specific transcription factor activities in cadmium-exposed animals. This research will broaden our understanding of the pathogenic mechanisms by which environmental agents act to elicit disease, should contribute to our understanding of individuals that are especially sensitive to toxicant immunomodulation, and may suggest new avenues of therapeutic benefit in Cd and other toxicant-exposed patients.
DESCRIPTION (provided by applicant):
The long term goal of our research is to reveal the molecular and cellular basis of sensory neural activation by hazardous environmental irritants. Sensory nerve endings in the lung, skin, eyes and mucous membranes are activated by environmental toxicants, including the alpha, beta-unsaturated aldehydes. Aldehydes such as acrolein (2-propenal) are severely irritating volatile compounds present in cigarette smoke, smoke from fires, automobile exhaust, and smog. In humans, acrolein exposure has been implicated in the pathogenesis of acute lung injury, chemical hypersensitivity, chronic pulmonary obstructive disease (COPD), and asthma. Despite its important role in human exposure health effects, the molecular targets of acrolein and other environmental irritants on sensory neurons remain unknown.
Here, we propose to identify and characterize the molecular targets for acrolein and other environmental irritants on somatosensory neurons. Our specific aims are derived from our preliminary data that show that acrolein, and a different class of irritants, the isocyanates, activate neurons by stimulating Ca2+ influx through the ion channel TRPA1. TRPA1 recently has been identified through its sensitivity to allyl isothiocyanate (mustard oil), an irritant used to probe pain transduction pathways. We found that TRPA1 is exclusively expressed in sensory fibers that are also sensitive to capsaicin, the plant-derived irritant that activates the capsaicin receptor, TRPV1. These sensory fibers, the C-fibers, are known to mediate irritant effects and inflammatory pain.
The objective of our application is to investigate whether TRPA1 is underlying acute and long-term irritant effects of acrolein and other environmental exposures in vivo. Our specific aims are to 1) investigate sensory neural responses to acrolein in TRPA1-deficient mice, 2) to elucidate the mechanism of activation and inflammatory sensitization of TRPA1, 3) study and compare the effects of industrial isocyanates on sensory neural activity, and 4) examine the effects of hazardous environmental toxicants on sensory neural activity and sensory neural receptors. Our experimental approaches will include the generation and analysis of transgenic mice, microscopic imaging of neural function, electrophysiological techniques and genetic and biochemical approaches.
DESCRIPTION (provided by applicant): Methylmercury (MeHg) is a persistent environmental toxin that selectively disrupts development of the fetal brain. Human exposure to MeHg, which occurs predominantly through fish consumption, contributes to an estimated 6% of women of child-bearing age in the U.S. having blood mercury levels at or above the reference dose set by the EPA. Children born to mothers having elevated blood mercury levels show cognitive deficits. Despite the ongoing health risks posed by MeHg the discrete mechanisms that make the developing nervous system most sensitive to MeHg toxicity are not clear. As well, factors that confer resistance to MeHg intoxication are not fully understood. In this proposal we will investigate how MeHg interferes with the earliest events in neurogenesis. Our overall hypothesis is that MeHg acts specifically in the nervous system by overcoming endogenous defense mechanisms to alter activity of cell signaling pathways. Using the Drosophila (fruit fly) model, we have discovered that MeHg can activate Notch receptor signaling, a highly conserved pathway required for normal neurogenesis in flies and humans. We also find overall resistance to MeHg is achieved by upregulation of glutathione synthesis specifically in the nervous system. We will therefore investigate how these fundamental mechanisms operate to disrupt, and alternatively protect, nervous system development with three Aims. First, we will characterize three distinct cell differentiation events in embryonic neurogenesis where Notch signaling is potentially perturbed by MeHg. Second, we will identify and characterize direct interactions of MeHg with protein targets in the Notch pathway. Finally, we will identify gene products that confer MeHg resistance by artificial selection and expression profiling. These data will advance our understanding of the fundamental molecular mechanisms dictating the susceptibility of the embryonic nervous system to MeHg toxicity.
DESCRIPTION (provided by applicant): Bisphenol A (BPA) is an estrogenic chemical that is widely used in the manufacture of policarbonate plastics and epoxy resins. Because BPA leaches out of plastic food and drink containers, including baby bottles, as well as the BPA-containing dental prostheses and sealants, considerable potential exists for human exposure to this compound. We have demonstrated for the first time that treatment of ovariectomized female rats with BPA dose- dependently inhibits both the hippocampal synaptoplastic and the positive cognitive effects of estrogen administration. These effects of BPA can already be observed at a dose of 40 ¿g/kg that is below the current U.S. Environmental Protection Agency reference daily limit for human exposure. Moreover, several publications have also raised the same worrysome issue that low- dose BPA might compromise normal sexual development and function of the brain. However, the majority of these studies, including our own, investigating BPA effects on the brain have been performed in rats. Hence, the implications of these alarming results to human health are intensively debated. Therefore, we propose to reveal the effect of BPA on estrogen-induced spine synapse formation in the brain of nonhuman primates, which will provide more relevance to human health. There is ample evidence that remodeling of synaptic contacts might mediate the beneficial effects of estrogen on both hippocampus- and prefrontal cortex-dependent executive functioning, particularly working memory. Thus, we hypothesize that BPA exposure may interfere with estrogen-induced synaptogenesis in the hippocampal CA1 and CA3 areas and dentate gyrus, as well as in the prefrontal cortex of nonhuman primates. This hypothesis will be tested using ovariectomized, adult young vervet monkeys that will be treated with vehicle- (controls), estrogen-, BPA- and estrogen+BPA. Furthermore, regarding the receptor target(s) of BPA, we will test whether BPA interferes with the positive effects of estrogen on the hypothalamic expression of oxytocin (O) and the progestin receptor (PR). Neurons producing PR and O contain different estrogen receptors, ERa and ER¿, respectively. Light and electron microscopic unbiased stereological calculations will be used to determine the total number of spine synapses in the hippocampus and prefrontal cortex, and the number of hypothalamic PR- containing and O-producing neurons. Bisphenol A (BPA) is an estrogenic chemical that is widely used in the manufacture of policarbonate plastics and epoxy resins. Because BPA leaches out of plastic food and drink containers, including baby bottles, as well as the BPA-containing dental prostheses and sealants, considerable potential exists for human exposure to this compound. We have demonstrated for the first time that treatment of ovariectomized female rats with 40 ¿g/kg BPA, which is below the the current U.S. Environmental Protection Agency reference daily limit for human exposure, inhibits both the hippocampal synaptoplastic and the positive cognitive effects of estrogen administration. This study, has been performed in rats. Thus, the implications of these alarming results to human health are intensively debated by the chemical industry. Therefore, we propose to reveal the effect of BPA on estrogen-induced spine synapse formation in the brain of non-human primates, which will provide more relevance to human health.
DESCRIPTION (provided by applicant): Humans and other species have suffered adverse health effects thought to result from exposure to environmental chemicals that interact with the endocrine system. A significant portion of these effects have been manifested as decreased reproductive potential - often through their effects in utero leading to abnormal development of the reproductive tract. In humans and rodents these effects have been clearly demonstrated with diethylstilbestrol (DES). HOX genes are highly evolutionary conserved and impart segmental tissue identity during the development of undifferentiated body axes. We have demonstrated that HOX genes play an essential role in reproductive tract development. In the prior funding period we have shown that HOX gene expression is altered in mice after in utero exposure to DES, methoxychlor or bisphenol A. We will continue to use DES as our model endocrine disrupter due to its well characterized effects, however will also include these additional agents in selected experiments. The objective of this proposal is to test the hypothesis that the mechanism by which endocrine disrupters affect the development of the reproductive tract is by altering the epigenetic regulation of HOX gene expression. While the initial regulation is mediated through the estrogen receptor (ER a or (b), persistent defects in HOX gene expression after exposure to endocrine disrupters suggests epigenetic alteration of HOX expression. In this application we will determine the molecular mechanisms that regulate HOXA10 and HOXA11 as well as identify epigenetic modifications that regulate HOX genes in both mice and humans. First we will characterize the 5' and intronic regulatory regions of HOXA10 and HOXA11 and identify transcription factor binding sites. Based on preliminary data, we hypothesize that these regulatory regions are methylated in response to xenoestrogen exposure. We will identify the potential impact of DES induced methylation on transcriptional regulation of these genes. Second, we will define the epigenetic changes that lead to persistent Hox gene alteration in the absence of continued exposure in mice. Finally, we will determine if the molecular mechanism by which endocrine disrupters alter HOX gene expression in mice is conserved in humans. We will examine uterine tissue from women with known in utero DES exposure. We have previously shown that Hox genes are necessary for reproductive tract development and that altered Hox expression leads to developmental or functional alterations. Here we expect to demonstrate that endocrine disrupters alter HOX gene expression and will determine the molecular mechanisms that mediate this regulation. No good model exists to explain the mechanism of both the acute and long term diminished HOX gene expression following endocrine disrupter exposure in utero. We hypothesize that the effects are maintained long after exposure through epigenetic mechanisms such as methylation of HOX genes.
DESCRIPTION (provided by applicant): Ambient fine particle matter (< 2.5 |j,m in diameter [PM2.s]) is hypothesized to be a factor in the exacerbation of asthma. The current US EPA standard for fine particles may not protect a sensitive sub-group-children with asthma. Limitations in the few cross-sectional and panel studies of the health effects of particle exposure are numerous and include: small subject numbers, narrow range of asthma severity, narrow time frame of observation, no control for household characteristics and allergic status, limited information on particles (chemical speciation and sources), and poor control of gaseous co-pollutants. We recently completed a prospective, 1-year follow-up study of 466 physician-diagnosed asthmatic children residing in southern New England, a region that includes an area of non-attainment of the EPA standard. Preliminary analysis using data from our Childhood Asthma Study and using limited, publicly available data on chemical constituents of PM2.5, suggests that daily respiratory health is compromised by exposure to particular components of PM2.s. In this grant application, we propose to acquire and analyze the stored, fine-particle collection filters from EPA monitoring sites in our region for trace elements and elemental carbon. We further propose to take advantage of the 158,000 subject-days of symptom and medication use data from our Childhood Asthma Study to examine the impact of exposure to PM2.5 and PM2.5 constituents on respiratory health. The primary health outcomes are: mother-reported daily respiratory symptoms (wheeze, persistent cough, chest tightness and shortness of breath) and medication use; and monthly asthma severity, assessed using the Global Initiative for Asthma (GINA) guidelines. Associations will be assessed with longitudinal repeated measures analyses using generalized estimating equations as well as empirical Bayes methods that will take into account sources of variability of exposures. Gaseous co-pollutants, meteorological variables and potential confounders (season of the year, atopic status of the child, local traffic density) will also be included in the evaluation. Relevance: Results of this study will provide important information necessary to assess the adequacy of the EPA PM2.5 standard, could identify the component(s) of PM2.s having the strongest impact on respiratory health, and could lead to more cost-effective strategies for source-targeted air-pollution interventions.
DESCRIPTION (provided by investigator): Preterm delivery, intrauterine growth restriction and low birth weight are major causes of infant mortality and severe morbidity in the United States. We propose to investigate the hypothesis that maternal exposure during pregnancy to ambient air pollution (CO, NO2, O3,, PM2.5, PM10, SO2) and traffic (a significant local source of air pollution) is associated with increased risk for low birth weight (<2500 gm), preterm delivery (<37 weeks gestation) and small for gestational age birth (< 10th percentile weight for gestational age). Several studies have examined the relationships between these adverse birth outcomes and maternal exposure to ambient air pollution but results have been inconclusive. A major impediment to this research has been the large sample size needed to investigate these relationships as well as the level of personal information needed to address potential confounders and accurately estimate exposure throughout pregnancy. This study will utilize two related datasets for the same geographic area and time period: (1) an existing cohort of women (N=10,524) followed prospectively throughout pregnancy by the Yale Center for Perinatal, Pediatric and Environmental Epidemiology; and (2) birth certificate data (480,000 singleton live births in CT and MA, 2000 to 2006). The cohort data provides well characterized variables to control for all major confounders and information about each of the mothers' residences throughout pregnancy to accurately assess exposure. The birth certificate data provides the statistical power to investigate severe, less frequent outcomes (very low birth weight <1500 gm, very preterm delivery <32 weeks), and to examine the effects of air pollution and traffic among African American women, already at risk for preterm delivery and low birth weight. A comparison study will also be conducted to determine the reliability of birth certificate data for use in air pollution research. Since all exposure assessments have some limitations, we propose four methods to measure exposure to air pollutants: central site monitors; a GIS/traffic model; land use regression to measure NO2 exposure; and satellite imagery to measure PM2.5 exposure. The proposed study will have the power to estimate odds ratios of 1.15 and 1.25 in the birth certificate and cohort data, respectively. If an association is confirmed, reductions in specific types of air pollution may result in a reduction in adverse birth outcomes. PUBLIC HEALTH RELEVANCE Preterm delivery and low birth weight are major causes of infant death and disability in the United States. This study will investigate whether a mother's exposure during pregnancy to air pollution and traffic (a significant local source of air pollution) is associated with increased risk for these adverse birth outcomes. If confirmed, reductions in specific types of air pollution may reduce these major infant health problems.
DESCRIPTION (provided by applicant): Exposure to aeroallergens and air contaminants is hypothesized to be a major factor in the exacerbation of asthma. Results of our ongoing studies suggest that nitrogen dioxide (NO2) associated indoors with gas appliance use and outdoors with motor vehicle emissions, at levels well below the EPA Air Quality Standard, may be associated with respiratory symptoms in children at risk for developing asthma and with the exacerbation of asthma in asthmatic children. Our data are also suggestive of an interaction between indoor aeroallergens and NO2 concentrations in enhancing respiratory symptoms in infants and asthmatic children. The potential impact is great since 57% of homes in the US use natural or LP gas, traffic volume is increasing and virtually all homes have significant dust allergen levels. We propose a prospective epidemiologic study of 1,533 children with active asthma (5 to 10 years of age), to test the hypothesis that carefully quantified NO2 concentrations associated with indoor sources and vehicle traffic on state and interstate roads are associated with asthma severity. In addition, we will determine if home levels of NO2, in the presence of common indoor dust allergens (Der p 1, Der f 1, Bla g 1, Pel d 1, Can f 1 and fungi), are associated with an increased risk of asthma severity in children sensitized to those allergens. The above hypotheses will be evaluated while adjusting for factors known or suspected to increase risk of more severe asthma, including household and school characteristics. The study population will be drawn from elementary schools in 16 Connecticut towns using a modified version of the ISAAC questionnaire to identify children with active asthma. Initial risk factors will be assessed in a home interview, and asthma severity (symptoms, frequency of ER visits, medication use, lung function.) will be assessed prospectively for one year using telephone interviews administered quarterly. At the home visit, dust samples will be collected for allergen and fungi determinations and blood samples collected for antigen specific IgE determinations. Indoor and outdoor NO2 levels will be obtained quarterly for each home. Global Information Systems in combination with road vehicle density data will be used to assess the impact of traffic on asthma severity and outdoor NO2 concentrations at the home. Household gas appliances and traffic-related pollution may represent important environmental exposures for asthmatic children, for which effective interventions can be developed to reduce asthma morbidity.
DESCRIPTION (provided by applicant): Sensitive detection of proteins and bacteria is of central importance in biomedical research, with applications to elucidating disease pathways, immunology, medical diagnostics, systems biology, functional proteomics, and new drug development. Highly selective and sensitive binding-assay arrays are increasingly important in light of emerging areas of biomedical science and intensified world bioterrorism. Sensitive, rapid, specific, multiple detection of proteins, bacteria and toxins in biological and environmental samples is critically important for public health. The broad, long term goals of this proposal are to develop highly sensitive micro- and nano-biosensor arrays for proteins and bacteria using patterned single-walled carbon nanotubes (SWNTs) and biomolecule recognition elements. Major specific applications include simultaneous measurement of suites of protein biomarkers in serum for early cancer detection. Single walled carbon nanotubes (SWNTs) with approximately 1.4 nm diameters have the world's highest conductivity per unit mass and have excellent potential for electrical sensing of biomolecules. We have assembled approximately 30 nm long carbon nanotubes standing in 20-200 nm diameter bundles called SWNT forests with electrical links to conductive surfaces. These high surface area, patternable, conductive nanostructures provide new opportunities for highly sensitive biosensor arrays. In this project, we combine nanotube electrical transduction with specific molecular recognition of antibodies for proteins and bacteria. SWNTs as molecular wires will shuttle electrons vectorially between enzyme labels at their tips and external electrical connections. We focus here on peroxidase-linked amperometric immunoassays, in which enzyme catalyzed reduction of H2O2 provides electrical signals transmitted via SWNTs. In preliminary work, we attached antibodies to SWNT forests and achieved detection of several antigens in the ng/mL range and below. Using newly developed conjugates of nanotubes, peroxidases and secondary antibodies (Ab2) with high label-to-Ab2 ratios with the SWNT immunosensors, we recently detected prostate specific antigen (PSA) in human serum at 0.01 ng/mL (0.25 Fmol/mL), a detection limit better than the best commercial assays for PSA. Ultra-low NSB combined with conductive polymer wiring of nanotube junctions, along with multi-enzyme label strategies, promise the highest possible sensitivity. Thus, we will optimize the new Ab2 nanoconjugates, explore conductive polymer wiring strategies, and eliminate non-specific binding (NSB) of interferences via competitively adsorbed protein/surfactant additives and synthesis of novel low NSB organic surfaces on nanotube ends. Finally, we will integrate optimized fabrication/detection approaches into multi-analyte sensor arrays utilizing patterned SWNT-antibody assemblies. In addition to achieving our specific aims, this project should provide fundamental guidance for fabrication of sensitive microarray devices for a wide variety of biomedical applications.
DESCRIPTION (provided by applicant): Occupational exposures are estimated to account for 9-15% of adult asthma cases. Among the most common causes of occupational asthma are isocyanates, reactive chemicals necessary to make polyurethane products (construction, automotive, military, medical, foam, coatings, adhesives). Millions of people are currently employed in industries that utilize isocyanates, and the work-force at risk for exposure continues to grow along with consumer demand for polyurethane. Recognition and diagnosis of isocyanate asthma can be challenging for both patients and physicians. Symptoms can be insidious and difficult to associate with the workplace, especially delayed responses, which are often mistakenly attributed to environmental triggers. In workers that develop immunologic hypersensitivity to isocyanate, long-lasting decrements in lung function may occur, and persist years after exposure ceases. Exposure is the only known risk factor for isocyanate asthma, and is the major target for disease prevention. Contemporary industrial hygiene technologies (respirators, "spray booths") reduce, but cannot eliminate workplace exposure. New cases of isocyanate asthma continue to occur in an estimated 5 - 10 % of those occupationally exposed, and represent a substantial public health problem. Despite the well-known allergenicity of isocyanates, occupational exposure assessment and disease screening is limited. Diagnostic assays that reliably differentiate sensitivity to isocyanate vs other asthma triggers are greatly needed for clinical practice as well as workplace screening. Biomarkers of isocyanate exposure are also needed to help guide and monitor industrial hygiene efforts. We hypothesize that isocyanate serology assays can be developed as a strategy for disease surveillance and industrial hygiene (exposure) monitoring to help protect individuals who are occupationally exposed to isocyanate. The serodiagnostics that would be developed in this project are new in two major ways. (1) They would be based upon novel "isocyanate antigens", produced using a mixed phase (vapor/aerosol/liquid) exposure system developed by our laboratory, which mimics the air/liquid interface of the bronchial fluid that lines human airways. (2) They would utilize biomarkers of allergy (IgE) to detect hypersensitive individuals, AND make use of immunologic markers of isocyanate exposure (IgG), to help target and monitor industrial hygiene efforts. The final product of this project would be a sensitive and specific ELISA kit, incorporating these unique qualities, which would have widespread value for clinicians, research scientists, industrial management and health care providers. Published findings from our laboratory predict the ELISA kit will be far superior to the only 1 other similar product currently available commercially (by a single supplier that dominates the market), and will dispel false conclusions that may have been drawn from previous studies using isocyanate antigens of questionable biologic relevance. PUBLIC HEALTH RELEVANCE: This grant application focuses on developing a better understanding the allergenicity of isocyanates, a major cause of occupational asthma world-wide. The expected product of the study will be a serodiagnostic assay, based upon novel isocyanate-protein complexes, that can be used to protect workers health by (1) identifying hypersensitive workers, (2) identifying exposure hot spots in the workplace (3) monitoring internal exposure and (4) monitoring the effectiveness of industrial hygiene efforts.
DESCRIPTION (provided by applicant):
The proposed research aims to address critical gaps in scientific knowledge of ozone and health with a national assessment of ozone and mortality and hospital admissions through epidemiological studies and biostatistical analysis. Specifically, the primary objectives are to investigate the relationship between ozone and mortality and hospital admissions, with respect to mortality displacement, longer-term exposure, the shape of the distributed lag curve, potential interaction with particulate matter (PM), heterogeneity among community-specific estimates, and threshold effects. Recent synthesis of scientific information on ozone and health by the United States EPA revealed considerable gaps in the literature on ozone and human health, including those outlined in the specific aims. This research directly relates to the NIEHS goals of reducing the burden of environmentally associated disease, contributing to career enhancement of young researchers, and providing a sound scientific foundation to support policy. More than 100 million people in the United States reside in areas exceeding the ozone standard, making ozone one of the nation's most pressing air pollution problems. A key component of the proposal is to investigate potential interaction between ozone and PM through the development of interaction models and through study of whether variation in PM composition can explain spatial and temporal heterogeneity in ozone effect estimates. This work is consistent with NIEHS initiatives to identify the health effects from complex mixtures and to develop statistical and mathematical models that accommodate complex mixtures. Results from this research are anticipated to be highly policy-relevant, both with respect to ozone and in the long-term with regards to air pollution and health more generally. An overall objective of this work is to contribute statistical techniques that could be applied broadly in environmental health research, such as to other health outcomes and other pollutants. Relevance to public health: This research investigates how hospital admissions and mortality are affected by ozone, a common urban air pollutant. The goal of this work is to provide policy-makers, scientists, and the public with a better understanding of how ozone affects health in order to aid more effective decisions regarding protection of human health.
DESCRIPTION (provided by applicant)
The overarching goal of this competing renewal K24 Mid-Career Investigator Award in Patient-Oriented Research is to enhance training in clinical multidisciplinary research focused on environmental health at Yale, building upon the substantial progress made during the initial K24 award. The candidate, Carrie A. Redlich, MD, MPH, is a highly respected and productive clinical investigator with unique training and research experience in multidisciplinary environmental/occupational health research. This proposal will enable Dr. Redlich to expand her current research efforts focused on understanding the health effects of isocyanate exposures, enhance her own research skills, and greatly advance her mentorship program. The research plan includes currently supported multidisciplinary studies investigating the effects of exposure to hexamethylene diisocyanate (HDI) in auto body shop workers, risk factors, mechanistic pathways and preventive approaches. New research specifically supported by this K24 award will focus on diphenylmethane diisocyanate (MDI), the most commonly used isocyanate worldwide. It builds upon the substantial progress to date and a long-standing relationship with Uretek, a fabric coating company in New Haven that uses MDI in the production of polyurethane coating fabrics, and will address important questions concerning the biologically relevant antigenic form of MDI, the role of skin exposure, exposure-dose relationships, and host factors. The specific aims are:
AIM 1) Identify and characterize biologically relevant antigenic forms of MDI.
AIM 2) Evaluate the respiratory and health status of individual workers at Uretek.
AIM 3) Characterize individual MDI skin and respiratory exposure patterns at Uretek.
AIM 4) Characterize the effects of MDI exposure on gene and protein expression profiles using DNA microarray and proteomic analyses.
AIM 5) Integrate exposure, clinical, immunologic, physiologic, and demographic data with genomic and proteomic data to explore exposure disease associations and host susceptibility factors which modify risks of exposure. Importantly, this new project will also serve to advance Dr. Redlich's research skills in new areas and provide a unique opportunity to mentor junior.
DESCRIPTION (provided by applicant): The long term goal of this project is to identify the molecular mechanisms involved in the activation of sensory neurons by unsaturated aldehydes, particularly acrolein.
-Specific Aim 1. Investigate the responses of TRPA1-/- mice to unsaturated aldehydes
The physiological and behavioral responses of both wild-type and TRPA1 knockout mice to acrolein and other unsaturated aldehydes will be studied. The mice will be tested for differences in thermal and mechanical sensitization following application of the irritants.
-Specific Aim 2. Identify the mechanisms of acrolein activation of TRPA1 channels
Electrophysiological, biochemical and molecular techniques will be employed to determine the mechanism of acrolein activation of TRPA1 channels. An acrolein activation model will help reveal whether TRPA1 is agonized by other unsaturated aldehydes with similar mechanisms of activation.
-Specific Aim 3: Analysis of the role of TRPA1 activation during acrolein-mediated bladder cystitis
To investigate the interaction of TRPA1 channels with unsaturated aldehydes in internal organs we will use the cyclophosphamide cystitis model. Injection of the chemotherapeutic agent cyclophosphamide leads to the metabolic generation of acrolein that is accumulated in the bladder. Acrolein induces hemorrhagic bladder cystitis through activation of sensory nerve endings in the bladder wall. Induction of this condition in TRPA1-/- mice and wild-type mice will allow for the examination of the role of this channel in the inflammatory process. Behavioral, morphological and physiological studies will be conducted.
TRPA1 is a novel cation channel involved in sensory activity and nociception. The aims within this proposal outline experiments that will examine the molecular mechanisms involved in TRPA1 activation by acrolein. Deciphering the mechanisms of TRPA1 activation is relevant to many health related issues. Acrolein is a common environmental irritant as well as a metabolic product of oxidative stress and the targets for most of these compounds on sensory neurons remains unknown. Therefore, the results within this study can provide insight into how a sensory neural channel links an environmental irritant to a biological response.
Laborers-AGC Education and Training Fund (Laborers-AGC) is applying for the Hazardous Materials Worker Health and Safety Training cooperative agreement, RFA-ES-04-005 to conduct a Hazardous Waste Worker Training Program (HWWTP - $24,169,912) and a Hazmat Disaster Preparedness Training Program (HDPTP - $2,341,200), at a total cost of $26,511,112 over the five years of the program. Laborers-AGC will train approximately 23,000 trainees under the HWWTP and 2,060 trainees under the HDPTP. Laborers-AGC will use 24 fixed training sites and 2 mobile units to train workers who are, or have the potential to be, employed on hazardous waste sites, who are at risk of exposure to hazardous waste on the job, or who may provide assistance as Skilled Support Personnel (SSP) on disaster sites. Workers will learn to work safely in highly hazardous environments. The trained SSP will learn how an emergency response operation functions, understand the roles of SSP and other participating responders, and obtain an awareness of potential problems and appropriate mechanisms for their resolution. Laborers-AGC's training will meet job and regulatory requirements and help protect worker health and safety during the entire remediation or emergency response. Training includes hazardous waste worker (exceeding OSHA and NIEHS Minimum Criteria requirements)
and other environmental remediation courses, health and safety training and specific job skills courses as required to meet regional and specific job site needs. Training incorporates hands-on exercises, activity based instruction, and advanced training technologies. This nationwide training program targets Construction Craft Laborers; other building trades; and environmental contractor and government personnel. Special outreach and training efforts will focus on recruitment and retention of minority and non-or limited-English speaking workers. Environmental Preparatory, General Construction, and Life Skills training along with social support services provided by community based organizations will help disadvantaged workers succeed in training and job retention.
DESCRIPTION (provided by applicant)
Laborers-AGC Education and Training Fund (Laborers-AGC) is applying for the Hazmat Training at DOE Nuclear Weapons Complex, RFA-ES-04-006 to conduct a DOE Hazardous Waste Worker Training Program (HWWTP) at a total cost of $12,204,871 over the 5 years of the program.
The Laborers-AGC DOE HWWTP will train workers engaged in activities related to hazardous materials and waste generation, removal (including demolition and decontamination [D&D] projects), containment, and emergency response within Department of Energy facilities. The DOE HWWTP will provide workers with the necessary skills to work safely in hazardous environments present on DOE remediation projects and during potential emergency situations at DOE. |n addition, the program will promote the Integrated Safety Management system used by DOE; incorporate proven, activity based learning techniques; and support the continuous advancement of worker health and safety. Training includes hazardous waste worker training(exceeding OSHA and DOE requirements); DOE approved and contractor accepted radiological worker training; and other environmental remediation training, health and safety training and specific job skills training as required and requested to meet job-specific tasks. Training will incorporate hands-on simulated exercises, activity based instruction, and the use of advanced training technologies.
Laborers-AGC will use 8 regional training sites and 2 mobile training units to provide training for approximately 20,000 trainees and 500,000 contact hours. Continuation of the existing DOE HWWTP will ensure immediate program initiation and will reduce training and set-up costs. This nationwide training program targets Construction Craft Laborers; other D&D workers; DOE personnel; and DOE contractor employees.
DESCRIPTION (provided by applicant): The long-term objective of the research described in this application is to determine the physiological and clinical relevance of soluble epoxide hydrolase (sEH) polymorphisms in humans. Numerous studies have shown that sEH is involved in the metabolism of exogenous and endogenous epoxide substrates. Exogenous substrates include mutagenic and carcinogenic epoxides found in our diet. Endogenous substrates include fatty acid epoxides, which regulate vascular tone, and sEH knockout mice have significantly lower blood pressure than do wild-type mice. sEH activity in humans varies by more than 500-fold, however, the mechanisms that underlie this variability remain unknown. Preliminary studies described in this application reveal the existence of at least 6 amino acid variants in the human sEH protein. Based on heterologous expression experiments, we show that at least 2 of these variant amino acids significantly alter the specific activity of the human sEH protein in vitro. These results suggest that sEH activity variability in humans may be partially due to genetic polymorphisms in the sEH gene, and that these polymorphisms may influence an individual's susceptibility or response to exogenous or endogenous epoxides. Based on these preliminary results, the specific aims of this application are to: 1. Construct and further characterize human sEH variant proteins using a heterologous protein expression system, 2. Compare sEH genotypes with sEH enzyme activity and amount in human liver samples, and, 3. Compare sEH genotypes in a group of hypertensive and normotensive humans. These specific aims are designed to test the hypothesis that polymorphisms in the human sEH gene result in an altered sEH phenotype in vitro and in vivo. This work will provide a basis for understanding the mechanistic roles of sEH polymorphisms in determining human disease progression as related to exogenous and endogenous epoxide metabolism.
Crisp Terms/Key Words: hypertension, genotype, genetic polymorphism, human tissue, epoxide hydrolase, liver, human genetic material tag, enzyme activity, clinical research, protein quantitation /detection
DESCRIPTION (provided by applicant): Sensory nerve endings in the skin, airways, eyes and mucous membranes are activated by a wide range of hazardous chemicals. These include pulmonary chemical agents as well as vesicants and noxious industrial chemicals. Activation of sensory nerve endings in the nasal passages and airways induces pain, irritation and apnea. Extended chemical exposure promotes injury by causing the release of inflammatory neuropeptides that contribute to edema formation in the lung and skin, ocular damage, mucus hypersecretion, pulmonary obstruction, and blister formation. In animal models these effects can be strongly reduced by prior neural desensitization or denervation. Thus, pharmacological blockade of neural activation, as well as block of neuropeptide receptors in target tissues, could represent effective measures to prevent and alleviate the deleterious effects of pulmonary agents and vesicants. The molecular targets for pulmonary agents and vesicants on sensory neurons are largely unknown. Recently, a new class of sensory chemoreceptors, the TRP ion channels, was discovered. The founding member, TRPV1, is the receptor for capsaicin, the pungent and inflammatory ingredient in chili peppers that is used as an incapacitating agent. TRPA1, a novel TRP ion channel, is activated by mustard oil (allyl isothiocyanate), a potent irritant and neuroinflammatory agent. We found that TRPA1 is exclusively expressed in sensory fibers that are also sensitive to capsaicin. Mustard oil and other plant-derived activators of TRPA1 show intriguing structural similarities with sulfur mustard. In addition, we found that TRPA1 is strongly activated by hazardous industrial chemicals. These include a,p-unsaturated aldehydes such as acrolein, a pulmonary agent used in the First World War. Our functional and behavioral analysis of mice deficient in TRPA1 showed that TRPA1 is essential for sensory neural responses to mustard oil, acrolein and other chemical hazards. Thus TRPA1, and potentially other TRP channels, may represent effective targets to prevent and treat the toxic effects of pulmonary agents and vesicants. Our specific aims are to investigate the role of TRP channels in the response to pulmonary agents and vesicants in vitro and in vivo and to analyze the efficacy of TRP channel blockers and neuropeptide antagonists in the reduction of warfare agent toxicity.
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