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Record Count: 63
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DESCRIPTION (provided by applicant): The long-term goal of the proposed research is to study the transcriptional regulation of the phase II drug-metabolizing and clearing enzymes by the xenobiotic nuclear receptor PXR. The phase II conjugating enzymes such as the UDP-glucuronosyltransferases (UGTs) function in concert with the oxidative phase I cytochrome P450 enzymes (CYPs) to eliminate xenobiotics such as prescription drugs and over-the-counter medications, and endobiotics such as bilirubin, steroid hormones and bile acids. The nuclear receptor PXR was initially identified as a xenosensor to regulate the expression of CYP genes via its binding to the PXR-response elements found within the mammalian CYP gene promoters. The identity of PXR as a species-specific xenosensor has been established by previous transgenic and gene knockout studies. Having established CYP genes as PXR targets, the presence of candidate PXR response elements in genes encoding the phase II UGT gene products raises the potential for a broader physiological function of PXR in xenobiotic response and clearance. However, whether UGTs are induced by PXR is unclear. To investigate UGTs as potential transcriptional targets of PXR, we propose to: (1) clone the UGT1A1 promoter and characterize its regulation by PXR; (2) examine the regulation of hepatic UGTs in mouse models bearing heightened (VP-hPXR transgenic), compromised (PXR knockout), or "humanized" (PXR knockout/hPXR transgenic) receptor activity; (3) examine the effect of altered PXR activity on bilirubin homeostasis; (4) examine the regulation of intestinal UGTs by PXR. If the regulation of phase II enzymes such as the UGTs by PXR proven to be true, we are toward establishing PXR as a master transcriptional regulator of the mammalian xenobiotic response. The results of these studies will provide novel elements in understanding the transcriptional regulation of UGT. It is anticipated that elucidation of the molecular basis of UGT regulation using the "humanized" mice will have great implication in human physiology and diseases. These include bilirubin and hormonal homeostasis, drug metabolism, and chemical carcinogenesis.
DESCRIPTION (provided by applicant): This is a very opportune time to study the genetic basis of complex phenotypes such as susceptibility to cancer. There are undoubtedly many genes that contribute to this phenotype and discovering their identities should lead to advances in cancer diagnosis, prevention, and even treatment. The mouse has always been the best experimental system for genetic analysis of complex mammalian traits, and with the sequencing of the mouse genome the utility of mouse models for identification of the genes at cancer susceptibility/resistance loci is further enhanced. Using a transgenic mouse model, we have previously demonstrated that the ornithine decarboxylase (Ode) gene is a susceptibility gene for nonmelanoma skin cancer in the mouse. When Odc is overexpressed in the target cells for carcinogens in skin, a previously resistant mouse strain becomes highly sensitive to skin tumorigenesis. Despite the strong effect of Odc on susceptibility, this phenotype can be modified by genetic loci present in a variety of inbred mouse strains. Published and ongoing genetic mapping studies have identified two classes of modifier loci, those that affect total tumor multiplicity and those that affect predisposition to squamous cell carcinoma development. The genes present at these loci presumably function in important effector pathways that: (a) are relevant to the tumor development process and (b) sensitive to changes in intracellular polyamine levels caused by Odc overexpression. In this proposal, we will focus our attention on fine mapping of these modifier loci, identifying the genes at these loci, and developing new models to study the genes' function in vivo. The specific objectives are to: (1) Fine map and identify the gene at Moo1 a strong modifier of tumor multiplicity on chromosome 17; (2) Map an X-linked modifier locus in crosses involving the C57BL/6J and BALB/cJ strains; (3) Fine map and identify the gene at Moo2, a strong modifier of tumor multiplicity on chromosome 6; (4) Develop novel mouse models in which an important regulatory region of the human ODC gene is knocked in to the homologous region of the mouse gene.
Common allelic variants of modifier genes undoubtedly contribute greatly to the differences between individuals in human cancer susceptibility. Identifying such genes through research on human populations is extremely difficult, but utilizing genetic analysis in the mouse to identify mouse modifier genes is now quite feasible. In the very near future, it should be possible to test hypotheses about human cancer modifier genes using results derived from mouse models.
DESCRIPTION (provided by applicant): The Ah receptor (AhR) has been shown to be largely responsible for the toxic and tumor promotional properties of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), especially in rodents. In addition, the AhR is a key regulator of xenobiotic metabolism. The human population is exposed to low levels of TCDD and related compounds (e.g. planar PCBs), mostly through the diet. However, the actual long-term health effects of human exposure to TCDD remain to be fully elucidated. Little is known about the biochemical processes involved in the activation and regulation of this ligand-activated helix-loop-helix/basic region transcription factor. In this application the ability of the human and mouse AhR to regulate gene transcription through protein-protein interactions will be examined in three specific aims; 1) Use transgenic mouse models to compare the ability of the human versus the mouse Ah receptor to activate gene expression and mediate liver tumor promotion, 2) Determine the ability of the AhR to modulate transcriptional activity other than through binding to dioxin response elements (DRE), and 3) Determine whether the AhR can alter gene regulation as a monomer. To achieve these goals, a combination of AhR-null cell lines and null-AhR mice with a mutant form of mAhR, WT-mAhR or human AhR "knocked-in" will be used. An emphasis will be placed on determining the multiple mechanisms that lead to direct AhR-mediated changes in gene expression and the target genes that are affected, as well as examine liver toxicity upon exposure to TCDD. Significant preliminary data is provided to support each aim, including the production of the hAhR and mAhR-A78D transgenic mouse models. The combined results from these studies will establish how the AhR/ARNT heterocomplex mediates transcription through a DRE and a non-DRE driven mechanism, and whether differences in the structure of the human AhR results in altered modulation of gene transcription. Lay Relevance: Liver insufficiency can result from environmental and dietary chemical exposure and lead to death in humans. Results from these studies will determine the mechanisms used by the Ah receptor to alter gene regulation that can lead to liver toxicity or tumor formation.
Crisp Terms/Key Words: protein protein interaction, aromatic hydrocarbon receptor, neoplastic process, human genetic material tag, active site, environmental toxicology, tissue /cell culture, receptor binding, toxicant interaction, dioxin, liver, gene induction /repression, transcription factor, genetic transcription, molecular cloning, transfection, site directed mutagenesis, biological signal transduction, genetically modified animal, laboratory mouse
DESCRIPTION (provided by applicant): Peroxisome proliferators are an important class of tumor promoters that bind to members of the nuclear receptor superfamily named PPARs (Peroxisome proliferator-activated receptors). Several subtypes of PPAR have been discovered (alpha, beta, gamma), although the predominant liver subtype (PPARalpha) is our primary interest due to the critical role it plays in carcinogenesis. Little is known about the biochemical properties or post-translational mechanisms that regulate this important receptor. Thus, the central hypothesis to be tested is that PPAR( transcriptional activity is regulated by site-specific phosphorylation and entry into a dynamic chaperone complex containing hsp90 and/or XAP2 The central hypothesis and proposed specific aims are firmly supported by preliminary findings from our laboratories. Four specific aims are proposed: 1). Determine the sites of phosphorylation of PPAR( and examine their role in modulating receptor activity; 2) Examine the role of the co-chaperone protein XAP2 in the regulation of PPAR function; 3). Determine the mechanisms that regulate XAP2 expression; and 4). Examine the role of PPAR chaperones (hsp90, XAP2, hsp70, p23) on transcriptional regulatory complexes. The phosphoprotein PPARalpha is found in cytosolic extracts as a complex with hsp90 and XAP2; both proteins repress PPARalpha activity. This would suggest that regulation of PPAR( activity is quite complex. A series of in vitro and in vivo studies are proposed utilizing a wide variety of techniques including transgenic and knockout mice, immortalized cell lines, transient and stable transfections and protein biochemistry. Taken together the proposed studies will greatly enhance our knowledge of PPARalpha's mechanism of action and hence our understanding of an important class of carcinogen.
DESCRIPTION (provided by applicant): Eukaryotes dedicate hundreds (and in some cases thousands) of proteins towards the regulation of gene expression. Yet very little is known about how all of these proteins coordinate their behavior at the many thousands of genes that comprise a typical genome. Little is known about how this coordination changes as cells reprogram their genome in response to signaling events including environmental stress. The work proposed here uses Saccharomyces cerevisiae as a model cellular system to undertake a broad survey of where transcriptional regulatory proteins are located throughout the genome, and where they move to when the genome is reprogrammed by environmental signals, such as heat shock and other stresses. Heat shock provides a rapid and simple programming event for the cell. Preliminary studies on this project have already revealed novel insights into gene regulation by demonstrating that many genes undergo partial assembly of the transcription machinery at promoters. Partial complexes await signaling events that drive them into full assembly. The location of a wide range of proteins involved in transcription will be evaluated by chromatin immunoprecipitation assays in which microarrays are used to detected genome-wide binding events (so called chlP-chip). Location will be assessed under normal growth conditions and under a wide range of environmental stresses, with particular emphasis on heat shock. Relationships among binding events will provide new insights into transcription complex assembly and regulation. Additional mechanistic insight will be provided through genome-wide biochemical dissection of native transcription complexes isolated from cells. Our cells are constantly faced with environmental extremes, involving temperature, starvation, radiation and harmful chemicals. How we deal with this stress depends upon the action of our transcription machinery. Therefore, a broad understanding of how our transcription machinery works in the face of various stresses is essential for a physiological understanding of human health.
DESCRIPTION (provided by applicant):
People are exposed to radiation through various sources. The use of radiation for diagnostic and therapeutic purposes is increasing. While these procedures bring significant benefits, radiation exposure carries risks. Prediction of risk from radiation exposure can be improved by taking into account the different biological effects of low and high dose radiation and individual variation in radiosensitivity. Most of the data on the effect of radiation have been accumulated from atomic bomb survivors, the only large cohort of exposed individuals that has been carefully monitored. Genetic study of radiosensitivity is difficult because relatives rarely get exposed to the same type and doses of radiation. In this project, we will use a combined genomic and genetic approach to determine the mechanisms of cellular response to different doses of radiation and to identify genetic variants that influence radiosensitivity. We will use expression level of genes in irradiated cells as phenotypes. This allows us to expose cells to different doses of radiation and to obtain radiation-induced phenotypes from related individuals for genetic analysis. The specific aims are 1) determine and compare the gene expression phenotypes induced by 0.5 Gy and 3 Gy IR, 2) map the chromosomal regions that influence inherited variation in IR responsive genes in large families by linkage analysis, and 3) confirm the linkage results and narrow the candidate gene regions by association analysis, and functionally characterize candidate regulators of response to 0.5 Gy and 3 Gy IR exposures. The results will provide information on the molecular and genetic basis of individual response to radiation exposure and form a foundation for a personalized approach to risk prediction.
DESCRIPTION (provided by applicant):
Aging is characterized by loss of functional reserve, placing the elderly at increased risk of numerous diseases. Identifying the driving force behind this functional loss is essential for maintaining a healthy populace. Recent evidence from our lab and others implicates DNA damage as a cause of aging. This implies a strong environmental component to aging. The long term objective of this research is to understand the molecular mechanism by which DNA damage promotes aging. This coupled with identifying environmental causes of genotoxic stress will greatly facilitate prevention of age-associated diseases. ERCC1-XPF is an endonuclease required for repair of bulky monoadducts via nucleotide excision repair (NER) and DNA interstrand crosslinks (ICLs) via a distinct mechanism. Deletion of ERCC1-XPF in the mouse causes early onset aging. These mice therefore offer a unique, rapid and sensitive model system for discovering which genotoxins promote aging and how they do so. The phenotype of the Erccl mice cannot be attributed to loss of NER. Thus our working hypothesis is that rapid aging in ERCC1-XPF deficient mice is caused by their inability to repair ICLs and therefore a consequence of endogenous ICLs which are cytotoxic. To test this, the investigators engineered mice hypomorphic for ERCC1-XPF which age over the course of months, permitting interventional studies. These mice will be exposed to DNA crosslinking drugs and environmental agents that promote lipid peroxidation (LPO), a likely source of endogenous ICLs, to determine if these exposures exacerbate the progeroid symptoms of the mice. The investigators discovered a human progeria caused by mutation of XPF. Thus identifying the cause of rapid aging in ERCC1-XPF-deficient mice will have direct implications for human health. The specific aims of this project are: Aim I: To define the cellular response of ERCCl-XPF-deficient cells to DNA ICLs and LPO. ERCCl-XPF-deficient cells will be exposed to 8-MOP or angelicin, plant-derived psoralens. Photoactivation of 8-MOP induces ICLs and monoadducts, whereas angelicin produces only monoadducts. Cell survival, cellular senescence, apoptosis, mutation frequency and chromosomal aberrations will be measured. If our hypothesis is correct then, 8-MOP will be significantly more cytotoxic than angelicin under conditions where an equal number of DNA lesions are induced. ERCCl-XPF-deficient cells will also be exposed to cadmium, an environmental agent that promotes LPO, to determine if LPO elicits the same cellular response as ICLs. Aim II: To directly test the hypothesis that unrepaired DNA ICLs promote aging. ERCC1-XPF hypomorphic mice will be chronically exposed to the crosslinking agent mechlorethamine. A second cohort will be exposed to 2-chloroethylamine (which induces structurally related monoadducts but not ICLs) using a dose that induces the same number of lesions as mechlorethamine. If our hypothesis is correct, mechlorethamine, but not 2-chloroethylamine, will exacerbate the progeria in these mice. Results will be confirmed by comparing skin aging in response to topical 8-MOP versus angelicin plus UV-A in mice genetically deleted for ERCC1-XPF in the skin only. Aim III: To determine if lipid peroxidation (LPO) promotes aging in mice with defective ICL repair. LPO is caused by oxygen radical damage to membranes and yields products able to crosslink DNA. We hypothesize that LPO is a source of ICLs that contribute to the phenotype of the Erccl mice. LPO will be induced in ERCCl-XPF-deficient mice via exposure to CCL4 or cadmium. If our hypothesis is correct, Erccl mice will be hypersensitive to LPO compared to wild type mice and LPO will exacerbate their progeria. Results from these experiments will indicate if LPO promotes aging and if so, whether it does so by inducing DNA damage. These experiments will also reveal if two common industrial exposures promote aging.
DESCRIPTION (provided by applicant):
The long term objective of the proposed research is to understand the molecular mechanisms of genomic instability associated with aging and aging-related diseases. Accelerated telomeric DNA loss occurs in aging-related diseases and after exposure to some environmental DNA damaging agents. Lack of the DNA repair protein WRN accelerates telomere loss and causes the human progeroid Werner syndrome (WS) in which patients prematurely develop multiple aging-related pathologies. The specific goals of this proposal are 1) to determine the molecular mechanisms of telomeric DNA loss associated with DNA damaging agents and WS, and 2) to define the roles for the repair protein WRN in telomere preservation. The hypothesis to be tested is that damage directly to telomeric DNA contributes to telomere attrition, and that WRN protein preserves telomeres by preventing and/or repairing breaks in telomeric DNA. A shuttle vector mutagenesis assay that measures mutations in a defined target will be used to examine the role of WRN protein in preventing replication-induced telomeric DNA deletions that may occur either spontaneously or after exposure to the environmental mutagen chromium (VI). Exposure to Cr(VI) is associated with respiratory cancers, and induces replication-blocking adducts and breaks in DNA sequences that are prevalent in telomeres. Thus, Cr(VI) is an excellent model for investigating the consequences of environmental DNA damage on telomeric DNA replication. The shuttle vector approach allows for analysis of independent and rare mutation events and eliminates selective pressure against the loss of telomeric repeats. To investigate a role for WRN in repairing breaks at telomeres directly, confocal microscopy will be used to induce DNA double strand breaks selectively at telomeres with a UVA laser in live cells. The broad goals of this proposal are to determine the impact of DNA damage and environmental mutagens on the integrity of telomeric DNA in chromosome ends, and to examine cellular pathways for repairing telomeres. Given the critical role for telomeres in aging and cancer, a mechanistic understanding of the genetic and environmental factors that accelerate telomeric DNA loss should aid in the identification of risk factors for premature aging. Identifying mechanisms of telomere loss and cellular processes that preserve telomeric DNA is crucial for the design of intervention therapies that prevent or delay the onset of diseases associated with aging and cancer.
DESCRIPTION (provided by applicant): The main objective of this application is to determine whether the xenoestrogenic substances bisphenol A (BPA) and butyl benzyl phthalate (BBP) play a role in the initiation of human breast cancer, and if so, whether this effect is mediated by epigenetic mechanisms. The proposed study is based on the growing concern that estrogenic environmental compounds that act as endocrine disrupting chemicals might have potential adverse effects on hormone-sensitive organs such as the breast. This concern is further fueled by evidence indicating that natural estrogens, namely 17 ¿-estradiol (E2), are important factors in the initiation and progression of breast cancer. Therefore, the concern that BPA and BBP, which have estrogenic properties and are widely distributed in the environment, might also be carcinogenic for the human breast is well justified. For accomplishing these goals we will utilize our in vitro in vivo model in which we have demonstrated the carcinogenicity of E2 in the human breast epithelial cells MCF-10F. The utilization of this powerful and unique model will provide us a tool for exploring whether BPA and BBP have relevance in the initiation of breast cancer. Furthermore, we have found that the expression of E2-induced transformation phenotypes is associated with hyper or hypomethylation of genes controlling branching and ductulogenesis. These are the basis for our rationale to postulate that the xenoestrogens BPA and BBP can induce neoplastic transformation by behaving as epigenetic modulators inducing silencing of critical genes by hypermethylation and/or histone modification that lead to the initiation and progression of breast cancer. For this purpose, we propose the following specific aim: To determine whether the xenoestrogens BPA and BBP induce neoplastic transformation in human breast epithelial cells and whether the expression of transformation phenotypes is associated with epigenetic changes in genes controlling branching and ductulogenesis, and if this is the case, to determine if modifying their methylation status reverts the neoplastic process. To accomplish this aim we will use MCF-10F cells and primary cultures of human breast epithelial cells obtained from reduction mammoplasty. The cells will be treated with BPA and BBP using a protocol similar to that used for the treatment of these cells with E2, which will serve as a control. Methylation studies will be performed using Restriction Landmark Genomic Scanning (RLGS) and the identified genes will be further studied using methylation specific PCR (MSP) followed by confirmation of their expression and functional role. Altogether, these studies will provide first hand evidence on whether xenoestrogenic substances are able to induce neoplastic transformation in HBEC and that epigenetic mechanisms are involved in this process. Furthermore the manipulation of the methylated status and silencing of those epigenetically modified genes will provide not only an understanding of how these environmental contaminants are involved in breast cancer initiation but also will give us tools for developing preventive strategies to counteract their effect in the general population. RELEVANCE TO PUBLIC HEALTH: These studies will provide first hand evidence on whether xenoestrogenic substances like BPA and BBP are able to induce neoplastic transformation in HBEC and that epigenetic mechanisms are involved in this process. Furthermore the manipulation of the methylated status and silencing of those epigenetically modified genes will provide not only an understanding how these widely environmental contaminants are involved in breast cancer initiation but also for developing preventive strategies to counteract their effect in the general population.
DESCRIPTION (provided by applicant): Polycyclic aromatic hydrocarbons (PAHs) are environmental pollutants that have been upgraded to human carcinogens by the WHO, International Agency for Research on Cancer (IARC). PAH are airborne pollutants found in smoke from fossil-fuel combustion, car-exhaust, fine-particulate matter (PM2.5), and first and second hand tobacco smoke. Inhalation toxicity likely results in cancer of the lung and airway. PAH require metabolic activation to exert their deleterious effects, however, the major pathway of PAH activation in the carcinogen target cell, normal human bronchial epithelial cells (NHBE) is uncertain. Three pathways have been proposed: the radical cation pathway (P450-peroxidase dependent) which produces depurinating-DNA adducts; the diol-epoxide pathway (P450-dependent) which produces stable-DMA adducts; and the formation of reactive and redox-active o-quinones (aldo-keto reductase (AKR) dependent), which can give rise to covalent DMA adducts and oxidative damage of DMA. Based on the consistent over-expression of AKR isoforms and the loss of one allele of hOGG1 (human oxoguanine glycosylase the base excision repair enzyme specific for removal 8-oxo-dGuo from DMA) in patients with lung cancer, we hypothesize that AKRs activate PAHs in human bronchial epithelial (HBE) cells and this results in increased oxidative DNA damage. Four aims are proposed to test this hypothesis. In Aim #1, the metabolism of benzo[a]pyrene (BP) will be measured in parental H358 cells (transformed HBE cells) in the presence and absence of (P450 and AKR) induction and the levels of radical cation metabolites (BP-1,6-, 3,6- and 6,12-diones), diol-epoxide metabolites (BP-tetrols) and o-quinone metabolites (BP-7,8-dione) will be quantified and identified by LC- MS. In Aim#2, a stable isotope dilution method using [13C]-BP-metabolites as internal standards to monitor the BP-metabolome will be established. In Aim#3, NHBE cells immortalized with cdk4 and human telomerase will be used to monitor the BP-metabolome by LC-MS stable isotope dilution methodology in the presence and absence of P450 and AKR inducers. In Aim#4, the ability of BP, BP-7,8-dihydrodiol (AKR substrate) and BP-7,8-dione (AKR product) to cause AKR-dependent 8-oxo-dGuo formation will be examined in NHBE cells. These studies will identify major BP-metabolites in NHBE cells which could be used to biomonitor human PAH exposure, and validate candidate genes for genetic predisposition studies.
The Fox Chase Cancer Center in Philadelphia, Pennsylvania, the University of Alabama Comprehensive Cancer Center in Birmingham, Alabama, and the Mount Sinai School of Medicine in New York, New York, have joined efforts for organizing a Breast Cancer and the Environment Research Center. Researchers at the three participating institutions have a long-lasting commitment to breast cancer research, demonstrated through pioneer work that has identified critical windows of susceptibility to carcinogenic and environmental exposures. The unique experience of this multi-institutional and multi-disciplinary group will provide a broad and comprehensive armarmentarium encompassing varied populations, clinical and basic sciences, and bioinformatics expertise that will be immediately translated to the population it will serve through carefully orchestrated programs under the guidance of patient advocates. The Breast Cancer and the Environment Research Center proposed here will be named Center for the Study of the Environment and Mammary Gland and Development. The Center is organized around two research projects, the Collaborative Project 1 (CP 1) and Collaborative Project 2 (CP 2), and two cores, the Administrative Core and the Community Outreach and Translation Core (COTC). The Administrative Core will serve as a liaison with the Steering Committee of the Breast Cancer and the Environment Research Center Consortium, the Breast Cancer and the Environment Task Force and the National Advisory Board. In addition, there will be an Executive Committee, which will be chaired by the Principal Investigator and will be integrated by the Co-Principal Investigators and one Cancer Advocate. The Executive Committee will receive advice from the Internal and the External Advisory Boards. The Executive Committee will meet on regular basis with the Co-PIs and investigators participating in CP 1 and CP 2, as well as with Members of the Administrative Core and the COTC. Consequently, the chain of authority for the Center grant will start from the Principal Investigator who will provide scientific leadership for all aspects of the Center. These lines of authority will not interfere with the internal chain of authority at each one of the participating institutions. The mission of the Center will be accomplished through interactive meetings among the participants of the Center, and other centers participating in this consortium.
DESCRIPTION (provided by applicant)
This is a revised application to establish a NIEHS Center in Environmental Health Sciences at the University of Pennsylvania. The Center will hold the title: "Center for Excellence in Environmental Toxicology (CEET)". It will be the single entity that coordinates, facilitates, synergizes and integrates all environmentally related health research and outreach at the University. The CEET has identified 50 faculty members in 16 Departments from 5 Schools as Center members. These members are dedicated to the CEET mission, which is: to improve the health of the public living in urban environments with an aging industrial infrastructure, through basic, translational and clinical research, and outreach. The investigators will relate environmental exposures, to molecular/cellular effects, to health outcomes, with the vision that diseases with environmental etiology can be largely eliminated by effective prevention or intervention strategies and/or changes in environmental policy. The mission will be accomplished via four integrated Research Cores, three Facility Cores, and a vibrant Community Outreach and Education Program (COEP). The Research Cores are led by investigators with international reputations. These Cores are in Oxidative Stress and Oxidative Stress Injury (co-Directors: Drs. Blair and Ischiropoulos), Endocrine and Reproduction Disruption (co-Directors: Drs. Strauss and Manson), Lung and Airway Disease (co-Directors: Drs. Panettieri and Albelda), and Genes and the Environment (co-Directors: Drs. Rebbeck and Whitehead). These Cores address environmental health issues important to our urban region. Adverse reproductive and pregnancy outcomes, developmental disorders, and diseases of the lung and airway are common, and are addressed in the organ-based Cores. The Cores in Oxidative Stress and Genes in the Environment will elucidate key mechanisms by which environmental exposures can lead to toxicity in these and other organ systems leading to an integrated Center structure. The Facility Cores will be in Toxicogenomics, Toxicoproteomics and Biomarkers, and will provide investigators with services that would otherwise be unattainable and will propel investigators into the new era of predictive toxicology. The COEP will disseminate the research findings and utilize the expertise of CEET investigators: to enhance K-16 education in environmental health; to conduct community outreach in four environmentally challenged Southeastern Pennsylvania communities, and to enrich the education of health care professionals. The COEP has forged relationships with community partners for outreach. The COEP goal will be to reduce the risk of harmful exposures and improve the public health of vulnerable populations within these communities.
DESCRIPTION (provided by applicant): Although consistent associations between ambient PM pollution and cardiovascular disease have been identified, the exact mechanisms, the time course by which PM causes the toxic effects, and effects of different PM species are not adequately understood. This study investigates clinically relevant arrhythmogenic, cardiac autonomic control, repolarization, myocardial ischemia, blood pressure (BP), inflammatory, coagulation, and fibrinolytic mechanisms and time course of PM2.5 effects (both concentration and species). Specifically, is personal short-term exposure to elevated ambient PM2.5 directly related to: (I) the onset of arrhythmic episodes (2) higher frequency of arrhythmias (3) lower heart rate variability (HRV) indices? (4) prolonged repolarization measured by QT index? (5) ST-segment level? Can this association be totally or partially attributed to the change of HRV and QT? (6) elevated ambulatory BP (SBP, DBP, and PP)? What are the time-courses (from minutes to hours) of the effects listed from 1-6 above? (7) Is personal short-term exposure to elevated ambient PM2.s (24-hour cumulative exposure) directly related to elevated markers of inflammation, blood coagulation, and fibrinolytic activity? (8) Are 24-hour cumulative concentrations of the ambient PM2.5 species, mostly emitted/formed from coal-fire power plant and vehicle combustion, associated with the above endpoints (Hypotheses 1-7)? (9) Are the associations between ambient PM2.5 concentration and cardiac endpoints (Hypotheses 1-7) modified by the types of PM2.5 spec/es? (10) Is there a synergistic interaction between PM2.5and long-term cardiac risk in the above relationships? (II) Are persons with older age, diabetes, and hypertension more susceptible to the above PM2.5 effects? We will (1) Recruit 100 patients with only arrhythmias of moderate frequency; (2) Perform a battery of cardiac tests to determine cardiac structural and functional status; (3) Measure concurrently 24-hour Holter ECG, ambulatory BP, and personal PM2.5 exposure, and derive real-time PM2.5 concentration, EGG, and BP data on each participant; (4) Analyze filters to assess major PM2.5 species; (5) Collect pre- and post- monitoring blood samples to assess inflammation and fibrinolytic activities; (6) Assess long-term risks of cardiac events; and (7) use time-series and cross-sectional analyses (both multivariable linear regression, transition models) to test the above research hypotheses. This study requires multidisciplinary collaborations of environmental, cardiovascular, and biostatistics researchers. The results will allow us to better understand the mechanisms and time course by which PM2.5 affects the cardiac system, and identify factors that lead to differential susceptibility to PM, and guide regulatory agencies in designing air pollution control standards.
DESCRIPTION (provided by applicant): Chronic exposure to trivalent arsenic (As III) is well known to cause cardiovascular diseases. In the human liver, As (III) promotes vascular remodeling, portal fibrosis, and hypertension, but the molecular pathophysiology of these As III-induced vascular changes is unknown. In general, the pathogenesis of As III induced vascular diseases has been understudied, in part, due to a lack of relevant animal models sensitive to chronic low dose As III effects. Our preliminary results show that exposure of intact adult mice to as low as 10 ppb of As III in their drinking water caused defenestration and capillarization of liver sinusoidal endothelium (LSEC). Furthermore, we also show that primary, short term cultures of murine or human LSEC are useful in revealing functional roles for As (III)-stimulated NADPH oxidase (NOX) generation of reactive oxygen species in the early signaling events affecting phenotype (e.g. fenestration) of this important target cell. The objective of the proposed studies is to use these in vivo and ex vivo models to investigate the mechanisms through which As (III) initiates LSEC remodeling and the molecular pathology of As (III)-induced vascular diseases. The global hypothesis for these studies states that As (III) acts at the level of g-protein coupled cell signaling to promote NOX oxidant generation that disrupts maintenance of LSEC fenestrations and suppression of capillarization. Accordingly, the specific aims of this proposal are to determine: I. the molecular mechanism by which As (III) causes liver sinusoidal capillarization and remodeling in intact mice. Wildtype and NOX deficient (p47phox -/-) mice will be exposed to As (III) (10-50 ppb) for 2 weeks and morphometric (light and electron microscopic level) determinants of SEC defenestration and capillarization will be quantified. Pharmacologically (antibodies to vascular endothelial cell growth factor receptor or Pertussis toxin) modified mice will be used to assess the contribution VEGF receptor and Gi-protein linked signaling to As III-induced vascular changes. II. the role of NOX generated superoxide in mediating As III-induced phenotypic conversion of primary murine and human LSEC. LSEC isolated from human liver or wildtype and p47phox -/- mice will be exposed to As (III) ex vivo to demonstrate mechanisms through which an imbalance of reactive oxygen and nitrogen species generation mediates AsIII-dysregulation of VEGF receptor maintained LSEC fenestration. III. if an imbalance in LSEC GT Pase activity mediates As (III) stimulated remodeling of the LSEC. Targeted delivery of RhoA or Rac1-GTPase siRNA and selective GT Pase activity assays will be used to dissect the roles of Rho family members in As (III)-stimulated LSEC capillarization.
DESCRIPTION (provided by applicant): Prostate cancer is the most frequently diagnosed cancer and second leading cause of cancer death in the United States. Androgen receptor (AR) activity is required for prostate cancer growth, and therapeutic regimens for prostatic adenocarcinoma (CaP) are directed toward ablation of AR activity. Despite the initial efficacy of these therapies, recurrent tumors ultimately arise wherein AR has been inappropriately re-activated. No effective treatment exists for recurrent tumors, which lead to patient death. As such, there is an urgent need to identify the comprehensive set of factors that contribute to AR re-activation and tumor recurrence. We have shown that selected endocrine disrupting compounds (EDCs) prevalent in the environment can impinge on the most common mutant of AR that arises during tumor progression (AR-T877A) to stimulate receptor activity and concomitant cellular proliferation. Our new data demonstrate that this action of EDCs has clinical consequence, wherein a known EDC (bisphenol) increased tumor growth and shortened the time to tumor recurrence. Moreover, we showed that several tumor-derived AR mutants gain sensitivity to selected EDCs. Thus, these findings strongly support the central hypothesis of the present proposal, wherein secondary mutations known to occur during tumor progression can sensitize cells to EDC activity, and through these mechanisms EDCs can significantly impact the response to prostate cancer therapy. Here, we delineated 3 specific aims which challenge this hypothesis by first discerning the molecular mechanisms by which EDCs impinge on mutant AR function (Aim 1), identifying the comprehensive cohort of secondary alterations that sensitize cells to EDC activity (Aim 2), and challenging the biological consequence of EDC exposure in the presence of these secondary alterations using new models of disease (Aim 3). Combined, it is our belief that this proposal rigorously addresses the molecular and biological significance of EDC exposure on prostate cancer management and outcome. Given the persistence of each studied EDC in the American population, these studies may have significant impact on understanding the factors that impinge upon prostate cancer growth.
DESCRIPTION (provided by applicant): The liver X receptors (LXRs), including the a and B isoforms, are highly expressed in the liver. LXRs can be activated by endogenous, natural, and synthetic ligands. Previous studies on LXRs have been focused on their roles in cholesterol and lipid homeostasis and inflammation. However, whether or not LXRs play a role in the regulation of xenobiotic enzymes and transporters and consequently impact the xeno-and endobiotic responses is unknown. Our preliminary results showed that transgenic mice expressing the activated LXRa (VP-LXRa) had an altered expression of multiple Phase I and Phase II enzymes, and possibly drug transporters. The most notable is the induction of the Phase II sulfotransferases (SULTs) in the VP-LXRa mice and LXR agonist-treated wild type mice. These include SULT2A9, a bile acid detoxifying hydroxysteroid sulfotransferase, and estrogen sulfotransferase (EST, orSULT1E1), which catalyzes the sulfation and deactivation of the estrogens. Promoter analysis strongly suggested SULT2A9 as a transcriptional target of LXRs. We also showed that the LXR a and B double knockout (LXR DKO) mice had decreased basal expression of SULT2A9. SULT2A1, the human homolog of SULT2A9, was induced in LXR agonist-treated primary human hepatocytes, suggesting that the SULT2A regulation may be conserved in humans. The induction of EST/SULT1E1 in the transgenic mice was associated with an inhibition of estrogen-induced uterine epithelial proliferation and estrogen receptor target gene expression. Based on our preliminary results, we hypothesize that the mouse and/or human SULTs are transcriptional targets of LXRs. A testable prediction is that the LXR-mediated activation of bile acid-and estrogen-metabolizing SULTs will lead to the alleviation of bile acid hepatotoxicity and cholestasis as well as down-regulation of estrogen activity in vivo. By using the "gain-of-function" VP-LXRa transgenic, "loss-of- function" LXR knockout and LXR ligand-treated wild type mice, we propose the following specific aims: (1) To determine whether the activation of LXRs alleviates bile acid hepatotoxicity and cholestasis; (2) To determine whether the activation of LXRs can functionally deprive estrogens; and (3) To determine the molecular basis by which LXRs regulate the mouse and human SULT2As and ESTs/SULT1E1s. These studies are expected to reveal a novel function for LXRs in protecting xeno- and endobiotic chemical challenges. We propose that LXRs have evolved to have dual function in maintaining cholesterol and bile acid homeostasis by increasing cholesterol catabolism; and, at the same time, preventing toxicity from bile acid accumulation. It is anticipated that the development of selective LXR agonists may represent a novel strategy to prevent cholestasis and limit estrogen activity in vivo. Since estrogens are prerequisites for breast cancer, delineating the regulatory effects of LXRs on EST/SULT1E1 expression may have a broader significance in the understanding of breast cancer prevention and treatment.
DESCRIPTION (provided by applicant): There is a clear, but poorly understood, link between estrogen and the autoimmune disease systemic lupus erythematosis (SLE). Humans are commonly exposed to Bisphenol A (BPA), a component of epoxy resins and plastics such as polycarbonate, and BPA binds to estrogen receptors to induce physiological changes. SLE-prone NZB/WF1 mice fed a small dose of BPA comparable to that obtained after a single application of an epoxy dental sealant, show a delay of onset of SLE symptoms and altered cytokine profiles. SLE symptoms arise from tissue damage caused by autoantibody production. Antigen presenting cells (APCs) are necessary for the activation of CD4+ T cells, whose help is required for autoreactive B cell differentiation and subsequent autoreactive antibody production. Given the requirement for MHC-II antigen presentation by APCs in this system and the core role that lysosomal proteases, cathepsins, play in this process, we will investigate the regulation of cathepsins by estrogen and bisphenol A in normal (BALB/c and C57BL/6J) and SLE-prone (NZB/WF1) mice. Using Western blots and active-site directed biotinylated probes, we will establish if there are cathepsin expression or activity variations in APCs (B cells, dendritic cells, and macrophages) between normal and SLE-prone mice as well as between healthy mice and those with the disease. In addition, we will establish if cathepsins are regulated by estrogen and BPA through ex vivo exposure of APCs, and if the regulation varies between normal and SLE-prone mice. Moreover, in vivo regulation by BPA will be monitored in mice fed physiologically relevant levels of the compound. These investigations will provide basic information on the interplay between the endocrine and immune systems and the specific effect of this interplay on the autoimmune disease SLE. It will also clarify the role of the endocrine disrupting chemical BPA on a central class of enzymes.
DESCRIPTION (provided by applicant): Human exposure to various chemical or pollutants is associated with an increased incidence of autoimmune diseases. Nevertheless, there are only a few useful animal models to study the mechanisms of chemically induced autoimmunity. In genetically susceptible mice, subtoxic doses of mercury elicit a complex autoimmune syndrome with production of highly specific IgG antinucleolar antibodies and a polyclonal increase in serum IgG1 and IgE. Mercury exposure affects various components of the immune system and, in this proposal; we plan to characterize the effects of mercury on B cells. Our preliminary data lead us to propose the hypothesis that mercury accelerates B cell maturation in the periphery and that mercury triggers secondary immunoglobulin gene rearrangements. In Specific Aim 1, we will assess how mercury influences the development of peripheral B cell subsets. We are particularly interested in determining whether mercury administration results in the upregulation of RAG proteins and in secondary immunoglobulin rearrangements. In Specific Aim 2, we will examine how mercury affects the expression and regulation of an anti-DNA transgene that has been bred onto a mercury susceptible genetic background. In Specific Aim 3, we will generate mice that possess a site directed transgene for an antinucleolar antibody. We will characterize the regulation of these B cells and evaluate their responses to mercury exposure.
DESCRIPTION (provided by applicant): Porphobilinogen synthase (PBGS) is an ancient protein, essential to nearly all-cellular organisms. PBGS catalyzes the first common step in tetrapyrrole biosynthesis (i.e., porphyrin, chlorophyll, vitamin B12), which is the condensation of two molecules of 5-aminolevulinic acid to form porphobilinogen. Despite multiple crystal structures, the order and identity of the catalytic steps remains unclear. The PBGS family consists of metalloproteins among which the utilization of metal ions has a unique phylogenetic variation between Zn2+, Mg2+, and K+. Three hypotheses drive the proposed studies. First, we propose that there are mechanistic differences between the PBGS that use Zn2+ and those that do not. Second, we propose that there is a physiological significance to our newly observed hexameric form of PBGS. All previously deposited crystal structures show an octamer. Third, we propose that a subunit-to-subunit communication outside the active site is the structural basis for the half-site reactivity evident in PBGS. Four interrelated Aims address these hypotheses.
Aim 1 is directed at elucidating the human Zn2+-PBGS catalyzed reaction mechanism. One novel approach uses a variant designed to process two different substrates, instead of two ALA molecules. We propose to use kinetic techniques, isotope effect determinations, 13C and 15N NMR, and other collaborative approaches.
Aim 2 focuses on the newly discovered hexameric form of PBGS. A hexamer-octamer transition is proposed to be the structural basis for allosteric activation by Mg 2+ for some PBGS. We will also probe for a relationship between altemate quaternary forms of the protein and an allelic variation reported to predispose some humans toward the environmental disease of lead poisoning.
Aim 3 describes investigation of the Mg2+ requiring PBGS where we focus on mechanism, structure, and the quaternary structure equilibria.
Aim 4 focuses on Drosophila melanogaster PBGS, which is a superior system for the study of the Zn 2+ utilizing PBGS and an excellent model for probing the subunit-to-subunit communication required for half-site reactivity. In support of these aims are collaborations to probe the structure and function of PBGS using X-ray crystallography, Raman spectroscopy, and analytical ultracentrifugation.
Crisp Terms/Key Words: magnesium ion, active site, analytical ultracentrifugation, nitrogen, zinc, stable isotope, enzyme structure, enzyme mechanism, porphobilinogen synthase, X ray crystallography, Raman spectrometry, nuclear magnetic resonance spectroscopy, chemical kinetics, carbon, Drosophilidae
DESCRIPTION (provided by applicant): Liver cancer is the third leading cause of cancer deaths throughout the world. The major risk factors for development of this disease are environmental in nature. These risk factors include agents that strongly modulate tumor susceptibility such as chronic infection with hepatitis B virus or exposure to the DNA damaging hepatocarcinogen aflatoxin B1 (AFB1). Tumor development is largely driven by genetic lesions that subvert critical cellular pathways. 1 such pathway, the retinoblastoma tumor suppressor (RB) pathway is mutated in the majority of liver tumors. Although RB is mutated in most liver tumors, how RB loss specifically contributes to tumorigenesis in the liver is unknown. Furthermore, the interplay between RB-deficiency and exposure to the environmental agents that cause liver cancer, e.g. AFB1, and how this impacts tumor formation is unclear. This is a critical consideration because it is well established that RB is required for the appropriate cell cycle checkpoint response to DNA damage. In the absence of RB, cells continue to proliferate in the presence of DNA damage, inducing additional mutations and leading to genome instability, a hallmark of cancer cells. Critically, the role of RB in DNA damage checkpoints in animals and how checkpoint abrogation contributes to tumorigenesis has not been studied. We have generated mice that have hepatocyte-specific RB-deficiency. We have found that RB deficiency causes abnormal hepatocyte ploidy (total DNA content), suggesting that these cells are inherently genetically unstable. We have also found that RB loss predisposes to the induction of liver tumors following genotoxic damage. Thus, we hypothesize that RB loss enhances tumor formation in the liver following exposure to environmentally-relevant genotoxic carcinogens, such as AFB1. We propose to delineate how RB modifies the response to AFB1 in the mouse liver and probe how this contributes to tumor formation. First, we will determine how RB loss influences the acute checkpoint response of the liver to AFB1 exposure and identify features of this aberrant response (e.g. development of genome instability) that may be important for driving tumor formation. Second, we will specifically elucidate the effects of RB-loss on AFB1-induced liver tumor development in the mouse. These studies will identify critical facets of RB function in vivo and delineate specific consequences of RB loss that are germane to tumor formation following genotoxic insult.
Crisp Terms/Key Words: terminal nick end labeling, genetic susceptibility, gene environment interaction, DNA damage, aflatoxin, carcinogenesis, liver neoplasm, neoplasm /cancer genetics, environment related neoplasm /cancer, immunocytochemistry, gene mutation, tumor suppressor gene, cell growth regulation, genetically modified animal, laboratory mouse, diethylnitrosamine
DESCRIPTION (provided by applicant): The Early Autism Risk Longitudinal Investigation (EARLI) Network will establish and prospectively follow a cohort of mothers of children with an autism spectrum disorder (ASD) at the start of a subsequent pregnancy (with select preconception data collection). This enriched-risk cohort will have the potential to serve as a study population for a variety of observational studies on the etiology and natural history of ASDs. The Network includes an Administrative Center at the Drexel University School of Public Health, a Data Coordinating Center at University of California Davis, a Central Laboratory and Repository at the Johns Hopkins School of Public Health, and four field sites: 1) Drexel University School of Public Health / Children's Hospital of Philadelphia; 2) University of California Davis / MIND Institute; 3) Johns Hopkins University School of Public Health / Kennedy Krieger Institute; and 4) Northern California Kaiser Permanente. The EARLI Network will implement a core epidemiologic data collection protocol focusing on: 1) prospective documentation of exogenous exposure during pregnancy and early life; 2) collection and banking of biologic samples during pregnancy and early-life, allowing measurement of exposure biomarkers, endogenous risk biomarkers, genotypes, and gene-expression profiles; and 3) follow-up of the newborn sibling through 36 months of age to gather data on ASD diagnoses, continuous ASD behavioral domains, and other potential behavioral endophenotypes. The goal is to have data on 1,000 siblings of ASD probands from fetal life through 36 months of age. This will allow sufficiently-powered estimation of associations among exposures and biomarkers measured during the pre-, peri-, and neonatal periods and ASD risk as well as candidate gene, gene-environment interaction, epigenetic, and maternally-mediated genetic effects. Full data collection in the target sample size will require additional five-years beyond the initial award period; however, by the end of the award the Network will have followed 875 pregnancies and completed 250 24-month follow-up visits. The attached application describes a plan for establishing the cohort and presents a set of exemplary specific aims to be partially addressed during the initial award period and more completely addressed once data collection is completed. Other types of specific aims that could be addressed through additional analyses of EARLI Network cohort data are highlighted.
DESCRIPTION (provided by applicant): Neurodevelopmental disabilities in children from low-to-middle income countries produce a substantial negative impact on societal and economic growth. Many of the causes of developmental disabilities, such as environmental toxins, nutritional deficiencies, psychosocial stressors, are common in resource-poor countries. Young children are particularly vulnerable to environmental exposures because rapid development creates windows of opportunity for toxins to exert their effects. In areas of the world where little research on neurodevelopmental disabilities has been conducted, it is necessary to assess the prevalence and nature of cognitive and behavioral deficits. In Montevideo, Uruguay, there is evidence that children are exposed to multiple heavy metals. However, to date very little research exists in this unique setting to understand the effects of multiple metal exposures on cognition, behavior, and school performance of urban children. There is also no research on how these exposures could be prevented or mitigated. We propose to conduct an investigation of cognitive performance and behavior in 200 children aged 6-7 years from Montevideo, Uruguay. Early school age is a period of important intellectual and social growth, and it sets up children's scholastic trajectories. At this time, learning disabilities often become manifested and diagnosed in children. It is also an age when environmental insults are clearly manifested. The goal of this project is to build research capacity and infrastructure in environmental epidemiology. Also, it is to investigate the relationship between heavy metal exposures (lead, arsenic, mercury), trace metals (iron and manganese) and cognition, behavior, and school performance in children from Montevideo, Uruguay. In the process of collecting data, we will identify key steps in the research process that will require additional training or resources. PUBLIC HEALTH RELEVANCE: Neurodevelopmental disabilities in children from low-to-middle income countries produce a substantial negative impact on societal and economic growth. Many of the causes of developmental disabilities, such as environmental toxins and nutritional deficiencies are common in resource-poor countries. We will conduct an investigation of cognitive performance and behavior in 200 children aged 6-7 years from Montevideo, Uruguay to understand the complex relationship between heavy metal exposures (lead, arsenic, mercury), trace metals (iron and manganese), and cognition, behavior, and school performance in children.
DESCRIPTION (provided by applicant): The developing brain is a particularly vulnerable target for lead with developmental lead exposure resulting in cognitive and motor deficits that persist into adulthood. Although the effects of lead on the developing brain have been studied for decades, there are gaps in our understanding of how genetic background and environment may modify lead's influences on nervous system development and function. Different environmental milieus may have powerful effects on the response of the brain to lead. For example, environmental enrichment could have potential neuroprotective effects against developmental exposure to lead whereas an impoverished environment may exacerbate the neurotoxicity. In addition to environment, genetic background may also modify the outcome from developmental lead exposure, although at this point, this has not been examined systematically. Thus, our proposed research has the following specific aims: Specific Aim 1. Examine the influence of genetic background on lead induced behavioral and molecular deficits in the hippocampus. Using gene expression arrays and bioinformatics, we will first survey the normal developmental gene expression profile for the hippocampus, a structure known to be sensitive to developmental lead toxicity in 4 strains of rats typically used for neuroscience or toxicology studies. We then will examine changes in these expression profiles following developmental lead exposure. We hypothesize that rats of different genetic backgrounds will have different behavioral and gene expression responses to similar lead exposures. Data derived from this study will lead to the first gene expression database for lead toxicity and advance our understanding of the metabolic, signaling and regulatory pathways that may be disturbed by developmental lead exposure. Information on the influence of genetic background on the response to this toxin may help in development of new strategies for intervention. Specific Aim 2. Assess the extent to which different environments modify behavioral and molecular deficits associated with developmental lead exposure. These studies will examine the effects of different housing environments on lead-induced deficits in spatial learning and memory and initial candidate gene expression (i.e., neurotrophic factor and NMDA receptor subtype expression) in the hippocampus. We hypothesize that animals raised in a social but impoverished environment will have more severe deficits and that animals reared in enriched environments and that the latter will be at least partially protected against the detrimental effects of lead exposure regardless of dose or type of exposure. Our research will not only examine the role of genetics in influencing the outcome from lead exposure but may demonstrate that the effects of lead on the brain are not immutable. Early intervention with therapy based on the principles of environmental enrichment might prove useful for attenuating at least some lead-related functional deficits. PUBLIC HEALTH RELEVANCE: The proposed research will provide new data on the role of genetics in influencing the outcome from developmental lead exposure by assessing how different types and levels of developmental lead exposure interacts with genetic variation to result in brain damage. Additionally, examination of how rearing in different environments modify behavioral and molecular deficits associated with development lead exposure may demonstrate that the effects of lead on the brain are not immutable and that early intervention with therapy based on the principles of environmental enrichment might prove useful for attenuating at least some lead-related functional deficits.
DESCRIPTION (provided by applicant): The primary hypothesis of this project is that organophosphate and pyrethroid exposure in utero and early childhood are associated with impaired neuropsychological development in the first three years of life. Data on actual levels of OP and pyrethroid exposure in children are scant, and there are no data on developmental effects at low doses. This is a cohort study of 440 infants enrolled at birth at the Magee Women's Hospital. At birth, subjects' meconium will be analyzed for organophosphate metabolites and pyrethroids, and umbilical cord blood will be analyzed for lead. Birthweight, length and head circumference will be measured at birth, and a neonatal neurological examination conducted. Children will be followed until 3 years of age. At each epoch appropriate developmental outcomes will be measured and measurements of OP's, pyrethroids and lead obtained. The effects of OP's, pyrethroids and lead on outcome will be measured, and interaction effects of the toxicants with OP's will be modeled. Other covariates to be evaluated are mother's age, education, IQ, socioeconomic status, smoking and alcohol intake. At present reference levels for OP's are derived solely from measures of Acetylcholinesterase in peripheral rodent blood. No developmental data on humans, either adults or children, are available. This project will address severe void in present day knowledge of children's exposure to pesticides, and the effects of these agents on neurobehavioral development.
DESCRIPTION (provided by applicant): Parkinson disease (PD) is caused by progressive and substantial loss of dopaminergic neurons in the midbrain. Although PD symptoms can be alleviated by dopamine replenishment therapy, the available treatments cannot protect the neurons at risk or prevent the disease from progression because we have a limited knowledge on PD pathogenesis. Recent advance in genetic and epidemiological studies suggests a multifactorial etiology for PD. Besides aging, genetic and environmental factors are associated with the risk for PD. In animal models, exposure to the environmental toxin paraquat causes permanent and accumulative damage to dopaminergic system. Most PD cases have no causative mutation in the identified PD genes, but there may be epigenetic modifications to neuron-protective genes during aging process. How PD risky factors interact to cause the disease remains to be elucidated. One possible pathogenesis for PD is that the disease is caused by early exposure to environmental toxicants in combination with subsequent dysfunction of neuron-protective genes during aging process: early exposure to environmental toxins may reduce the threshold of developing PD; later dysfunction of one or more neuron-protective genes may accelerate the neuronal death, cross the reduced threshold for PD onset, and result in Parkinsonism. The genetic models for PD are mostly generated by constitutive mutation of targeted genes and thus cannot model temporal dysfunction of a gene later in life. To test this hypothetic pathogenesis of PD, we have created conditionally mutated mice to produce temporal and partial dysfunction of the targeted genes after the animals are exposed to environmental toxin in their early lives. We will use paraquat as a PD-related environmental toxin and PINK1 as a PD-linked gene. Dopamine likely plays an important role in PD pathogenesis because disturbance in dopamine homeostasis causes severely pathobiological conditions leading to neuronal death. Therefore, we will study the neuron-protective gene VMAT2 since VMAT2 plays a major role in dopamine homeostasis. This proposal will test how early exposure to environmental toxin superimposes deleterious effect on phenotypic expression subsequent to disturbance in dopamine homeostasis in later life, subsequent to later dysfunction of the PINK1 gene, or subsequent to later dysfunction of two neuron-protective genes. Results from this study will advance our understanding of PD pathogenesis and provide guidance to the development of therapeutic strategies for treating this devastating disease. PUBLIC HEALTH RELEVANCE Environmental and genetic factors are associated with the risk for Parkinson disease (PD), but how these risky factors interact to cause the disease is unclear. A potential pathogenesis for PD is that the disease may be caused by early exposure to environmental toxins that reduces the threshold of developing PD and later dysfunction of one or more neuron-protective genes that may cross the reduced threshold to cause the disease. The genetic models of PD are mostly generated by constitutive gene mutation and thus cannot model temporal dysfunction of a gene later in life. We have created conditionally mutated mice as the models to test how early exposure to environmental toxin superimposes detrimental effects on phenotypic expression subsequent to later dysfunction of neuron-protective genes. Results from this study will advance our understanding on PD pathogenesis and provide guidance to the development of therapeutic strategies for PD treatment.
DESCRIPTION (provided by applicant): We seek to elucidate the impact of hypoxic stress on placental differentiation and function, and consequently fetal growth. An adequate supply of proteins, carbohydrates and fat is obligatory for fetal development. Transfer of these nutrients is regulated by a set of well-orchestrated signals, programmed by genetic and environmental cues. Whereas our understanding of placental import of proteins and carbohydrates has markedly advanced in recent years, the mechanisms that govern uptake, accumulation and trafficking of fatty acids in placental trophoblasts are largely unknown and insufficiently investigated. Recent data implicate the nuclear receptor PPARy, a master regulator of adipogenesis, bioenergetics and inflammation, in regulation of placental fatty acid uptake and transport. PPARY-null mouse embryos exhibit intrauterine growth restriction and subsequently fetal death, associated with diminished fat accumulation within the labyrinthine placenta. We have recently determined that PPARy enhances trophoblast differentiation, and stimulates trophoblast uptake of fatty acids as well as the expression of proteins that govern accumulation and trafficking of fatty acids in the placenta. Importantly, environmental insults, such as exposure to hypoxia, adversely impact placental function and are associated with intrauterine death or substandard fetal growth. The mechanisms underlying these injuries are unknown. Our basic and translational research approaches are designed to understand cellular and molecular underpinnings of trophoblast fat transport and metabolism, their regulation by PPARy and the impact of hypoxic stress on these processes. We utilize novel in vivo and in vitro approaches to interrogate previously unknown gene- environment interactions that determine synthesis, utilization and efflux of fatty acids in trophoblasts, and the molecular pathways that regulate the expression and function of pertinent fatty acid transporters. We pursue feedback circuits that are instigated by free fatty acid and influence triglyceride synthesis in human trophoblasts. Together, our research will unveil previously unrecognized links between placental lipid trafficking and hypoxic injury. Consequently, our studies are likely to pave the way to better understanding of placental dysfunction that culminates in fetal growth restriction and its life-long sequelae.
DESCRIPTION (provided by applicant): The objective of this exploratory research project is to study the effects of chronic exposure to phthalate esters on the infant primate testis in a novel xenograft model system. Environmental exposure to reproductive toxicants is of serious concern to human health and has been implicated in both the apparent decline of male fertility and a rise in testicular neoplasms. Phthalate esters are widely used as plasticizers in consumer products and can leach into the environment, resulting in significant human exposure, especially during childhood and in pediatric patients requiring long-term catheterization or intravenous treatments. Di-(2- ethylhexyl) phthalate (DEHP) is the most ubiquitous phthalate and its metabolite MEHP is a known testicular toxicant. Di-n-butyl phthalate (DBP) is commonly found in personal care products and fetal exposure to DBP affects male reproductive tract development in rats. Historically, research into the reproductive toxicology of phthalates has been evaluated following gestational exposure in rodents, but species-specific differences in metabolism and sensitivity of the reproductive organs suggest that results obtained in rodent models may not extrapolate well to humans. As epidemiological studies link phthalate exposure during childhood to disturbances of human male reproductive development, there is a clear need for studies in primate models, as emphasized by the NTP-CERHR Expert Panel on the Reproductive and Developmental Toxicity of DEHP. In this revised exploratory project we propose to utilize a novel primate testis tissue xenografting system developed in our lab to study the effect of DEHP and DBP on testicular maturation and testis function in immature rhesus monkey testes. Specific aim 1 will test the hypothesis that exposure to environmentally relevant concentrations of DEHP or DBP will adversely affect testicular somatic cell function and germ cell development. Specifically, testis tissue from infant and juvenile monkeys will be ectopically grafted to mouse hosts and exposed to various doses of DEHP or DBP for 3 months. At various time points, Sertoli cell maturation, steroidogenic function of Leydig cells, and exogenous gonadotropin-induced germ cell differentiation will be compared in treated and control tissue to assess the potential effects of chronic phthalate exposure. Specific aim 2 will investigate the mechanisms underlying phthalate toxicity in the infant primate testis. Phthalate-induced changes in global gene expression patterns in testis tissue from infant monkeys will be evaluated by microarray analysis and confirmed by RT-PCR. Special attention will be given to differential expression of genes involved in steroidogenesis. Successful completion of this exploratory project will provide insights into the sensitivity to phthalate esters, characterize effects on testicular somatic cell maturation, and begin to elucidate the molecular mechanisms underlying phthalate toxicity in the immature primate testis. Results from the proposed project will provide the basis for future studies performed in the developing primate. PUBLIC HEALTH RELEVANCE: Rodent models are conventionally utilized to study the potential effects of suspected reproductive toxicants, such as phthalates, on the immature human testis, but species-specific differences in metabolism and sensitivity of the reproductive organs may not make them ideal model systems. While studies in primates are more likely applicable to the human, they are often cost-prohibitive and ethically restrictive. In this research proposal we describe a novel system in which pieces of primate testis tissue are grafted into mice to investigate the mechanisms of reproductive phthalate toxicity in primate testis without extensive experimentation in primates.
DESCRIPTION (provided by applicant): Diverse stressors of chemical and microbial origin can produce lung inflammation and adverse cardio-respiratory health effects. Effects of single agents have been well described clinically and experimentally, however, we hypothesize that chemical and microbial stress can interact to produce a response that is greater than that predicted by the response to single agents alone. Metals, such as nickel (Ni) derived from atmospheric particulate matter (PM) are important components of ambient air pollution associated with adverse health effects. Mycoplasma are a class of microorganism that potentially can produce chronic/persistent/latent infections within the lung with minimal signs of infection. We hypothesize that the presence of microorganisms like mycoplasma will potentiate the inflammatory/immunomodulating potential of chemical stress and, thus, act as co-factors in the genesis or exacerbation of chronic inflammatory and fibrotic lung disease following exposure to PM-derived metals. We will utilize M. fermentans as a prototypic organism capable of latent/subclinical infection or colonization to deliberately infect human lung fibroblasts (HLF) in vitro in order to study the molecular and cellular basis for the synergistic interactions with residual oil fly ash and its attendant metals such as Ni on host-cell production of immune-modulating cytokines such as IL-6. We propose 4 specific aims: 1) To establish and characterize the synergistic interaction of chemical stress (ROFA and Ni exposure) and microbial stress (M. fermentans infection) to stimulate the production of inflammatory and immune-modulating cytokines by HLF 2) To demonstrate that toll-like receptor-2 (TLR-2) specific stimuli (MALP-2) and Ni synergistically interact to produce inflammation and fibrosis within the lung in vivo; 3) To define the importance of Jun-N-terminal kinase/stress-activated protein kinase (JNK/SAPK) in mediating the synergistic interactions between metal-containing Ni and MALP-2; 4) To determine if Ni exposure can enhance activation of innate immunity by microbial components via modulation of the early signaling events of TLR-2 signal transduction. These studies will provide valuable insight into the mechanisms by which microorganisms like M. fermentans upregulate host-defense mechanisms within the lung and modulate the inflammatory response to environmental chemicals. The identification of microbial agents as determinants of host-cell response to chemical stress will necessitate that microbial ecology be taken into consideration in the assessment of risk posed by atmospheric pollutants such as ROFA.
DESCRIPTION (provided by applicant):
Silicosis develops many years after initial exposure to silica particles and is characterized by chronic inflammation, lung fibrosis and ultimately respiratory impairment. There is no effective treatment for silicosis. As a result, patients diagnosed with silicosis are given a dismal prognosis and many patients are still dying of this disease. The biochemical mechanisms behind silicosis are poorly characterized, however oxidants do play a role in disease development. The antioxidant enzyme extracellular superoxide dismutase (ECSOD), the major antioxidant enzyme in the lung extracellular matrix (ECM), protects against other fibrotic lung diseases. Previous studies have correlated the loss of ECSOD with worsening lung injury and a pro-oxidant lung environment. ECSOD overexpression or treatment with SOD-mimetic drugs can prevent this injury. Thus, one component of an effective treatment strategy may be the restoration of the oxidant / antioxidant balance in the lung. Studies show that administration of mesenchymal stem cells (MSCs) can participate in the repair of lung tissue after oxidative injury. However, little is known about the effects of a highly oxidative lung microenvironment on stem cell-mediated tissue repair. Therefore, the investigators hypothesize that silicosis results in a pro-oxidant microenvironment that leads to the reduced efficacy of mesenchymal stem cells to repair lung damage. The investigators will employ a mouse silicosis model whereby MSCs purified from either ECSOD null mice or ECSOD overexpressing mice are administered to animals exposed to silica. The effects of modulating antioxidant levels of both the stem cells and the recipient mice will be determined on the efficacy of the stem cell treatment. When complete, these studies will provide valuable information on the influence of the lung microenvironment on the ability of stem cells to abrogate lung disease. These studies will also begin to address the feasibility of transplanting purified MSCs with augmented antioxidant capabilities into patients with silicotic lung disease as therapy. Lung disease due to silica exposure (silicosis) is a continuing public health problem that has no effective treatment. This project examines the use of adult stem cells along with increased levels of antioxidants in the treatment of silicosis. These studies will help determine the feasibility of using adult stem cells in the treatment of patients with silicosis.
DESCRIPTION (provided by applicant): Asbestosis, a form of pneumoconiosis caused by inhalation of asbestos fibers, is the prototype of diseases caused by inhalation of mineral fibers. There is currently no treatment for this disease. Pathologically, asbestosis is characterized by interstitial fibrosis in a pattern identical to that observed in patients with idiopathic pulmonary fibrosis (IPF) with the additional finding of asbestos bodies in histologic sections. The pathogenesis of asbestosis is not fully understood, but inflammation, reactive oxygen species (ROS), and increased expression of TGF-B all play important roles in this disease. Notably, the receptor for advanced glycation end products (RAGE) when activated by its ligands induces chronic inflammation, enhances ROS production and leads to TGF-B synthesis. Over the last decade, RAGE has been implicated as a progression factor in a number of diseases, including diabetic atherosclerosis, chronic inflammation and renal fibrosis to name a few. The role of RAGE in asbestosis has not been evaluated. However, in renal fibrosis RAGE signaling has been found to be a central mechanism in stimulating myofibroblast metaplasia. Importantly, myofibroblastic foci are believed to be key mediators of disease progression in fibrotic lung injuries such as asbestosis and IPF. Thus, RAGE signaling, which induces chronic inflammation, ROS production, TGF-B synthesis, and myofibroblast metaplasia has many features that suggest it may be a central mediator in the pathogenesis of asbestosis. In addition to the membrane RAGE just described, there is also a soluble isoform of RAGE (sRAGE) that has an extracellular (ligand-binding) sequence identical to that of RAGE, but lacks a transmembrane domain and consequently is secreted. sRAGE is thus a decoy molecule, and has successfully been used in animal models to stop progression of the diseases listed above. Notably, we have found that the lung contains extremely high concentrations of sRAGE compared to all other tissues, yet there have not been any studies of this protein in lung disease. Thus, sRAGE may have an important anti-inflammatory and anti-fibrotic role in the lung by preventing ligands from binding the membrane RAGE. In this proposal we will directly test the hypothesis that RAGE-mediated signaling is a central mechanism in the pathogenesis of asbestosis and that sRAGE can protect against this disease.
DESCRIPTION (provided by applicant): Zinc, a major soluble component of particulate matter, may be a critical toxic agent in the cardiorespiratory pathophysiologic effects of exposure to atmospheric dusts. Nonetheless, the molecular targets that underlie zinc toxicity within lung remain unknown. We recently reported that extracellular zinc (1-100 ?M) increased amiloride-sensitive sodium currents of heterologously expressed murine epithelial Na+ channel (ENaC) subunits. Since a) ENaC-dependent sodium reabsorption across airway epithelia has a significant role in regulating the volume of airway surface liquids (and hence mucociliary transport); and b) activation of ENaC is essential in the pathogenesis of chronic airway disorders such as cystic fibrosis, we hypothesize that activation of ENaC by zinc is an important molecular determinant of the toxicity of inhaled particulate matter. Accordingly, the specific aims of this proposal are (i) to determine the effects of zinc on ENaC activity and its impact on periciliary liquid (PCL) height, mucociliary transport (MCT) and ciliary beat frequency (CBF) in primary cultures of human bronchial epithelium (HBE); (ii) to determine the effects of zinc on ENaC activity in a heterologous expression system; and (iii) to identify the molecular mechanism by which zinc activates ENaC. Our proposed studies regarding the effects of extracellular zinc on ENaC activity in primary cultures of airway epithelium and in heterologous expression systems will provide fundamental biophysical and molecular information towards our understanding of the pulmonary response to inhaled particulate matter. Our proposed study will examine the link between excess zinc, a suspected major player in human lung injury due to inhaled particulates, and the activity of lung sodium channels that are critical regulators of mucus clearance, the primary lung defense mechanism. This study will enhance our understanding of mechanisms by which atmospheric dusts result in lung damage, and will provide basic information that may lead to the development of intervention measures.
DESCRIPTION (provided by applicant):
Polycyclic aromatic hydrocarbons (PAH) are ubiquitous environmental air pollutants that result from fossil fuel combustion and cigarette smoking. PAH exposure is a major risk factor in human lung carcinogenesis. A critical step in multi-stage carcinogenesis is the mutagenic event that results from the formation of DNA adducts. There are three principle routes of metabolic activation of PAH resulting in the formation of diol- epoxides, radical cations, or reactive and redox-active o-quinones. Each of these reactive metabolites have the potential to form DNA-adducts and these adducts may lead to mutation. We are using two approaches to model PAH carcinogenesis, a highly versatile yeast system and human lung cell lines. In lung cancer, the gene most often mutated is p53 where three distinguishing characteristics are found in lung cancer. (1) The pattern of mutations is dominated by G to T transversions; (2) The spectrum of mutations reveals hotspot codons, a few of which are unique to lung cancer; (3) The pattern and spectrum of mutations show a strand bias for mutations on the non-transcribed strand. Our systems use genetic selection methods so that change-in- function mutations can be detected with ease. The yeast reporter system for p53 relies on wild-type p53 binding to a promoter to drive an adenine reporter causing mutant colonies turn red. Our preliminary data suggest that PAH o-quinones, when permitted to undergo redox-cycling, generate 8-oxo-dGuo to cause predominantly G to T transversions with a modest, but significant, preference for hotspots but with no strand bias. We recently devised a system to detect mutations in human lung cells using a p53 dependent promoter to direct transcription of the Herpes TK gene. In this system p53+ cells are killed by exposure to ganciclovir while p53 cells are resistant. The mutant p53 is next rescued from the cells using the yeast system and then sequenced. Using these assays to model p53 mutagenesis we will: (1) Determine the role of repair genes on p53 mutagenesis and compare different PAH metabolites, (2) Map the locations of PAH induced DNA lesions in the p53 gene(3) determine if PAH-metabolites can mutate p53 in lung cells. Our hypothesis is that PAH o- quinones and the ROS they generate provide a route to the p53 mutations found in lung cancer. PUBLIC HEALTH RELEVANCE: Polycyclic aromatic hydrocarbons (PAH) are ubiquitous environmental air pollutants that result from fossil fuel combustion and cigarette smoking. PAH exposure is a major risk factor in human lung carcinogenesis. This proposal will study the mutagenesis of the p53 oncogene by PAH and their metabolites.
DESCRIPTION (provided by applicant): Published work points to an important role of surfactant protein A (SP-A) in innate host defense. SP-A modulates a number of host defense processes and exposure to ozone results in functional and structural alterations of SP-A. Two human genes, SP-A1 and SP-A2, and several genetic variants for each SP-A gene have been characterized. In vitro studies show functional, structural, or biochemical differences between the two genes and the gene-specific variants. SP-A levels are altered in a variety of lung diseases, and SP-A genetic variants are associated with risk for several pulmonary diseases. Our central hypothesis is that differences among SP-A variants account for differences in risk to lung disease in response to environmental insults. Our specific hypothesis is that the two human SP-A gene products are not functionally equivalent with regards to their host defense function, and that ozone exposure has a differential impact on this function. To investigate the specific hypothesis, we propose to study the effect of ozone, on pathogen-infected C57BL/6 and SP-A-/- mice (Aim 1) by assessing: a) survival with bacterial infection; b) ability to clear pathogens from their lungs and limit dissemination of infection; c) cytokine production, the in vivo phagocytic index of macrophages, and the in vivo oxidation status of SP-A; as well as, on the ex vivo host defense function of macrophages from wild type and SP-A-/- mice (Aim 2). To generate transgenic mouse lines on the SP-A-/- background that express equivalent levels of human SP-A1 or SP-A2 gene products (Aim 3). To study the impact of ozone on the SP-A1 and SP-A2 transgenic mouse lines by carrying out studies similar to those described for Aim 1 and by studying biochemical characteristics of the transgene products (Aim 4), and the ex vivo host defense function of macrophages from the SP-A1 and SP-A2 mouse lines (Aim 5). Through the proposed work we will generate an animal model to study human SP-A variants, and we will determine, in vivo, and in response to ozone exposure, the role of SP-A in host defense and assess functional host defense differences between the two human SP-A gene products. Knowledge gained may help explain and perhaps link the in vitro data with the human genetic association data, and provide insight in the understanding of the value of the SP-A gene duplication.
DESCRIPTION (provided by applicant): We feel airborne chemicals when they stimulate nerve endings in the eyes, nose, mouth, and throat. Government regulators view chemical irritation as a material impairment of health, and set many exposure limits accordingly. Basic data on sensory response to chemicals are vital for setting exposure-limits and for understanding the relationship between sensation and physiology. However, basic data are scarce, particularly in humans. Data on the effects of stimulus-duration are especially scarce. Sensory systems can integrate stimulus-energy over time to detect weak stimuli (temporal integration). Thus, one must study the domain of time to fully understand any system. Aim 1 a will examine integration in detection of nasal irritation for presentations that last up to several seconds. Short-term integration is important because much of the data on nasal irritation are based on brief exposures. For fixed concentrations, duration will vary to find the briefest pulse that causes perceptible irritation. So far, a simple but imperfect mass-integrator model fits plots of threshold-duration vs. concentration quite well. The experiment will test the hypothesis that simple but imperfect integration is common for other compounds. Aim 1 b will examine integration that occurs up to one minute, using pulsed stimulation with a rhythm similar to natural breathing. The research will test the hypothesis that integration occurs across "breaths," and can allow subjects to detect nasal irritaiton at lower concentrations than brief exposures would suggest. The studies will provide pilot data for more detailed studies that ask whether brief presentations can predict longer-term integration, such as might occur in natural environments. Aim 2 will test a new model of short-term integration based on transport of molecules. In the model, subjects perceive irritation when concentration in nasal tissue reaches a critical level. Concentration builds (integration occurs) as molecules diffuse into tissue from the air. However, diffusion out of tissue into the bloodstream undermines build-up. The model will be tested using using dynamic stimuli, i.e., pulses of irritants interupted by gaps of clean air. The model predicts that interrupting inflow into the mucosa will degrade detection by allowing diffusion out of the mucosa progress unchecked. The three experiments will lay groundwork for a psychophysics of dynamics that can help physiologists understand underlying mechanisms and policy-makers predict how our dynamic sensory systems will react over time.
Crisp Terms/Key Words: environmental exposure, odor, clinical research, sensation, stimulus /response, sensory mechanism, perception, olfactory stimulus, olfaction, respiratory system, pulmonary respiration, pulmonary diffusion, psychophysics, pollutant interaction, mathematical model, model design /development, human subject, irritation /irritant, time resolved data, air pollution
DESCRIPTION (provided by applicant): Silicosis remains a serious inflammatory lung disease for which no successful treatment is available. During the previous cycle of this grant we showed that patients with silicosis have poor response to lung transplantation. We also showed that silica promotes activation of p42 MAPK/ERK and induces phosphorylation of TNFR1, a process that modulates silica-induced cytotoxicity in macrophages. RAW 264.7 macrophages experience ERK-mediated phosphorylation of TNFR1 and are protected from apoptosis. In contrast, IC21 macrophages do not phosphorylate TNFR1, and experience enhanced apoptosis in response to silica. Silica induction of macrophage apoptosis is associated with mitochondrial dysfunction and the oxidation of cardiolipin (CL) that inversely correlate with TNFR1 phosphorylation. The mechanisms responsible for the silica-induced oxidation of CL are unknown but may involve the peroxidase activity of cytochrome c (cyt c). The mechanism(s) by which phosphorylated TNFR1 protects macrophages from silica-induced apoptosis and its effect on silicosis is unknown and constitutes the question addressed in this renewal application. We have assembled a team of researchers with expertise in TNF1 and lipid biology to study the importance of TNFR1 phosphorylation on silica-induced lung injury. Specifically, we will study the role of the intracellular TNFR1 in silicosis and its effects on caspase activation and mitochondrial damage. We hypothesize that following exposure of macrophages to silica TNFR1 undergoes phosphorylation and inhibits the formation of the pro-apoptotic signaling complex thus preventing apoptosis. Our central hypothesis is that phosphorylated TNFR1 inhibits the peroxidase activity of cyt C and limits the silica-induced oxidation of CL thus protecting macrophages from silica-induced apoptosis and ameliorating silicosis in mice. To test this hypothesis we propose the following specific aims: 1) To determine the mechanism(s) by which phosphorylated TNFR1 protects RAW 264.7 macrophages from silica-induced apoptosis. 2) To determine whether phosphorylated TNFR1 modulates the peroxidase activity of cyt c and reduces CL oxidation during silica-induced macrophage apoptosis. 3) To determine the consequences of in vivo expression of phosphorylated TNFR1 on silica-induced lung injury in mice. We will isolate macrophages from the bone marrow of C57BL/6 mice, to stably transfect them with viral constructs encoding for TNFR1 mutants to prevent or mimic TNFR1 phosphorylation. Subsequently, stably transfected macrophages will be transferred to silica-exposed mice to evaluate the effect of these cells on the development of silica induced lung injury in mice. PROJECT NARRATIVE: The term silicosis refers to the fibronodular lung disease caused by long-term inhalation of free silica. In the United States, more than 2 million people are exposed to silica every year and recent surveys indicate that a high percentage of these individuals are exposed to levels of silica above the permissible exposure limits. Subsequently, there were an estimated 3,600-7,300 newly recognized silicosis cases in the US from 1987 to 1996. Although preventive measures have decreased mortality attributable to silica the reality is that a large number of silica-exposed patients are still dying as a result of this disease. CDC surveys indicate that nine states, including Pennsylvania, report high rates of silica- induced mortality, more than 3 deaths/million people/year. These reports also point out that Western Pennsylvania exhibits a much higher mortality due to silicosis when compared to the rest of the country and clusters of counties (where mining is an important activity) around the city of Pittsburgh are identifiable that contribute disproportionably (> 14.8 deaths/million/year) to this mortality. Emphasizing the importance of these reports is our experience (described in our preliminary data) at the University of Pittsburgh with lung transplantation in patients afflicted with silicosis. We identified 12 patients that received lung transplantation for this disease from 1986-2004 . The outcome of these patients was contrasted with that of a group of 79 patients that received lung transplantation for IPF, a disease for which lung transplantation confers survival benefit. Our data indicate that patients with silicosis have poor survival rates (2.4 years) and experienced earlier rejection of their lung grafts (0.9 years) following transplantation than patients with IPF (5.34 and 2.4 years for survival and graft rejection respectively). Consequently, these data indicate that silicosis still is a serious and frequently lethal lung disease for which no successful treatment is available.
DESCRIPTION (provided by applicant):
ICx-Agentase aims to develop a continuously operating wearable badge that utilizes biocatalytic sensing elements to monitor chemical toxins in air. The proposed wearable, low maintenance, self-contained device will provide real-time measurements of chemical toxins at the point of contact and will provide wireless communication tools to forward the data to a central data collection station. ICx-Agentase is currently developing a wearable badge for detection of chemical agents with support from the United States Defense Threat Reduction Agency and the United States Army Research Office (W911NF-05-C-0004). At this point we have a fully functional, continuously operating prototype device for detection of nerve agents in air. This technology employs inexpensive sensing elements which need to be replaced periodically. The instrument does not require intensive maintenance or calibration. This technology has been successfully tested in independent live agent tests at an approved United States Government facility. With this three year research effort we aim to increase our detection capabilities and improve this prototype by making it smaller, lighter and cheaper to allow the large environmental population studies. Key features will be: High sensitivity with rapid response time (<2 minutes); Analyte specific quantitative detection (four sensing cartridges for detection of ozone, heavy metals, volatile organic compounds (VOCs) and pesticides, respectively); High resistance to potentially interfering environmental conditions such as temperature and humidity; High resistance to potential chemical interferents; Wearable with global positioning system (GPS) tracking and wireless communication to enable central data collection; Expandable platform technology (each device can support two interchangeable sensing elements for detection of two different exposure classes); Low maintenance; Inexpensive (< $5 operating costs per day - utilize replaceable sensing cartridges); Increase operational lifetime of sensing cartridges to at least 36 hours.
DESCRIPTION (provided by applicant): We have developed and prevalidated an in vitro screening test for phototoxicity, the Enhanced Phototoxicity Assay in Reconstituted Skin (EPARS), to replace the current standard animal phototoxicity tests. The phototoxic potential of chemicals, cosmetics, dietary supplements and Pharmaceuticals are a major and growing concern in the consumer products industry, but society and the government have demanded decreased use of experimental animals. EPARS is more objective, faster, and less expensive than animal tests. To-date, no alternative phototoxicity test has been validated in the U.S. (via ICCVAM), although the 3T3 Neutral Red Uptake (3T3NRU) assay has been accepted in the E.U. after validation by ECVAM. EPARS is significantly superior to the 3T3NRU viability test because it overcomes several of the 3T3NRU test's limitations. Specifically: (1) EPARS is based upon a differentiated tissue model that closely parallels human skin morphology, instead of a fibroblast monolayer; (2) the tissues are composed of primary human keratinocytes in a 3D culture system, a more relevant model than a mouse tumor cell line; and (3) test substances can be applied directly, avoiding the often problematic solubilization of formulations into culture media. If developed to its full potential, EPARS would be the most accurate assay available to identify potential phototoxic agents in humans. We propose to confirm the reproducibility, sensitivity, specificity and predictability of EPARS by testing an expanded list of known phototoxins and validate the most predictive and accurate tissue viability endpoints identified in Phase I. We will also use human gene microarrays to measure changes in gene expression patterns and identify genes that can be used as predictors for induction of phototoxicity. This Phase II work will supplement and strengthen the foundation of EPARS in preparation for submission to U.S. regulatory agencies as an alternative to animal phototoxicity testing.
DESCRIPTION (provided by applicant):
In responding to HazMat emergencies, practice not only makes perfect, it also saves lives. Under the Phase I SBIR grant, Amethyst Research developed a software simulation tool called HazCommand. Our Phase II goal is to create a model platform for credentialing operational competency of initial company officers responding to HazMat incidents. The credential will reflect the responder's ability to demonstrate operational competency that meets local operating procedures and NIMS/ICS and NFPA standards. By creating such a HazMat Incident Command System credentialing component for the Philadelphia Fire Department, we will have produced a concrete roadmap for other organizations to create their own standards-based credentialing program. Amethyst Research will accomplish this goal by 1) creating a comprehensive set of HazMat incident scenarios, 2) developing reliable evaluation metrics and competency- assessment credentialing processes, 3) developing support for metrics capture and reporting, and 4) evaluating the credentialing processes on representative sample. This project aims to mature a software simulation tool called HazCommand for training emergency responders in command and control, strategy, tactics, and communication, into a full-fledged learning platform. This project should impact public health by improving response by firefighters to HazMat incidents.
DESCRIPTION (provided by applicant): The long-term objective of this research is to develop powerful statistical methods for the analysis of data from genetic epidemiology studies. While voluminous data are becoming available owing to the Human Genome Project and rapid advancement of high throughput genotyping technology, powerful statistical methods are needed for ultimate success in identifying predisposing genetic variants and their environmental modifiers. This project focuses on developing statistical methods for analyzing genetic association studies on perinatal or early-life diseases. These studies very often adopt a retrospective case-control design, but they have a distinct feature in that offspring of mother cases/controls (for perinatal diseases) or parents of offspring cases/controls (for early-life diseases) are also recruited. Thus these studies have information on both unrelated case-control comparisons and genotype/haplotype transmissions within families. Another important feature of these studies is that the covariate distribution in the study population is structured so that genetic and environmental variables are usually independent within families. The fact that such independence does not hold in the case population under the alternative hypothesis provides further information on the association beyond standard case-control comparison. These studies usually seek to evaluate effects of both maternal and offspring genotypes/haplotypes, their interactions, and gene-environment interactions. Building on currently available approaches for analysis of case-control association studies and case-parent triads, we propose novel efficient estimation and testing methods that can account for the retrospective case-control design and incorporate the family information on the genotype/haplotype transmission and the structure in the covariate distribution. Classical logistic regression for case-control studies applies for most of the analysis but is less efficient due to the ignorance of family information and covariate structure. The Transmission/Disequilibrium type test or likelihood-based methods for analyzing case-parent triads discard the controls and/or their parents and cannot estimate all parameters of interest (e.g., main effects of environmental exposures). Our methods range from profile-likelihood methods and estimating-function based methods to hybrid methods based on the conditional likelihood for case triads and pseudo-likelihoods. This project is motivated by and will be applied to ongoing scientific studies at the University of Pennsylvania on which the PI is collaborating, and the phenotypes include pre-term birth, preeclampsia, hypospadias, and asthma. Our methods also have broad implications to the study of phenotypes other than perinatal and early-life diseases. We will develop large sample theories for the proposed methods, evaluate their finite sample performance by simulation studies, and demonstrate their usefulness using real data. Fully documented software to implement these methods for public use will be provided using freely available statistical package R. PUBLIC HEALTH RELEVANCE: This project proposes novel statistical methods for the analysis of data arising from case- control genetic epidemiology studies of perinatal or early childhood diseases. Data usually consist of case and control mothers and their respective offspring or consist of both case-parent triads and control-parent triads. The proposed methods are for the estimation and testing of maternal and offspring genotype/haplotype main effects and interactions and interaction effects between genotypes/haplotypes and environment variables.
DESCRIPTION (provided by applicant): The long term objective of this project is to develop powerful and computationally efficient statistical methods of identifying genes, environmental risk factors and their interactions underlying complex traits related to human diseases and health. The specific aim of this project is to continue to develop survival analysis models to incorporate age of onset data, environmental covariates information, gene-gene and gene-environment interactions into haplotype-based genetic association analysis, analysis of single nucleotide polymorphisms (SNPs) and admixture mapping of complex traits in population-based cohort studies. The project also evaluates different study designs in genetic association studies. The proposed methods build on our current methods and hinge on novel integration of methods in survival analysis, high dimensional data analysis and methods in human genetics. The focus will be on the development of rigorous and comprehensive statistical inference procedures for haplotype analysis, gene-gene and gene-environment interaction analysis and admixture mapping in cohort studies of unrelated individuals sampled by different study designs, including case-cohort and nested case-control designs. Likelihood-based inferences, hidden Markov models and threshold gradient descent methods will be developed for these aims. The project will also investigate the robustness, power and efficiencies of these methods. In addition, this project will develop practical and feasible computer programs in order to implement the proposed methods, to evaluate the performance of these methods through simulation and application to real data on breast and ovarian cancer risks among the BRCA1/2 carriers and to data sets in the area of pharmacogenomics. The work proposed here will contribute both statistical methodology to studying complex traits and methods for high-dimensional data analysis, and offer insight into each of the clinical areas represented by the various data sets to evaluate these new methods. All programs developed under this grant and detailed documentations will be made available free-of-charge to interested researchers via the World Wide Web.
Crisp Terms/Key Words: disease /disorder onset, linkage disequilibrium, gene environment interaction, human data, family genetics, spondylitis, rheumatoid arthritis, narcolepsy, computer assisted sequence analysis, mathematical model, genetic model, model design /development, data collection methodology /evaluation, linkage mapping, genetic disorder, celiac disease, insulin dependent diabetes mellitus, computer program /software, computer system design /evaluation, biotechnology
DESCRIPTION (provided by applicant)
Asbestosis is a debilitating disease caused by the inhalation of asbestos mineral fibers. Asbestosis is associated with chronic inflammatory reactions that can lead to the development of lung fibrosis years after the initial exposure. However, current treatment regimens are inadequate and new ones must be sought. It is well known that asbestos fibers generate reactive oxygen species in animal models of asbestosis, suggesting that oxidants play an important role in disease development. The enzyme extracellular supeoxide dismutase (ECSOD) is an antioxidant enzyme highly expressed in the lung and loss of ECSOD from the extracellular matrix (ECM) has been associated with pulmonary fibrosis in mice. ECSOD contains a heparin-binding domain that regulates its biodistribution in the ECM. This domain is sensitive to proteolysis, and removal of the heparin-binding domain may be an important mechanism regulating ECSOD levels in the ECM. ECSOD directly binds to type I collagen in alveolar septa, where it may protect collagen from superoxide-mediated fragmentation. Notably, collagen fragments are potent chemoattractants and activators of neutrophils. In addition, it has recently been shown that collagen fragments can induce apoptosis in a number of cell types. Therefore, any increase in superoxide production will accelerate both the inflammatory reactions and cellular destruction by initiating collagen degradation. The primary hypothesis to be tested in this proposal is that ECSOD protects against pulmonary fibrosis by preventing superoxide-mediated collagen degradation and apoptosis. To study this hypothesis, the applicant will pursue the following Specific Aims: 1) Investigate changes in ECSOD activity, biodistribution and sites of synthesis in mouse lungs at different stages of asbestos-induced lung injury, 2) Assess the structural effects of asbestos deposition on type I collagen, and 3) Determine the stimulus for and extent of programmed cell death in lungs of asbestos-treated mice.
DESCRIPTION (provided by applicant): This four-year FIC Development Award will foster the candidate's ability to achieve her long-term goal of becoming an independent interdisciplinary researcher. Ultimately she will develop the skills, knowledge and experience to pursue an international collaborative research career focused on global health challenges, particularly environmental and nutritional risk factors for child behavior problems. This goal will be achieved through involvement in a mentor-oriented international collaborative research study, through pursuing research activities, course work, laboratory training, and conferences/workshops. The proposed training plan aims to build on the candidate's prior research experience with new training in understanding the impact of environmental toxicity and / or micromalnutrition on children's behavior problems. The five specific training goals are to (1) expand new knowledge on environment toxicity, (2) receive further training in nutrition assessment and its implication in child behavior problems, (3) obtain further training in psychopathology, (4) enhance skills in research methodology and advanced statistics, and (5) develop new knowledge and skills in international collaborative research and cross-cultural research. The proposed research plan aims to examine the link between both environmental toxicity and micronutrition deficiency and childhood externalizing behavior. The four specific aims are to: (1) assess the direct effect of lead exposure and micronutrient deficiency on children's externalizing behavior; (2) examine the interactive effect of biological and psychosocial risk factors on childhood externalizing behavior; (3) test which factors mediate the above direct and interactive effects; and (4) identify the specific components of environmental toxicity/micronutrient deficiency and psychosocial risk factors which give rise to childhood externalizing behavior. Hypotheses will be tested in the context of an ongoing international collaborative prospective study of 780 five to six-year-old male and female Chinese kindergarten children. Measures include blood levels of lead and micronutrients, IQ, psychosocial confounds, and behavior problem checklists. Structural Equation Modeling will be used for data analysis. / In summary, this proposed mentored international collaborative research/ training will target three global public health problems: environmental toxicity, micromalnutrition, and childhood behavior disorder, a focus consistent with mission of FIC of addressing global health research priorities.
DESCRIPTION (provided by applicant)
Exposure to pesticides has been associated with oxidative stress and Parkinson's disease. Although different pesticides produce a common phenotype, the initial pathways of intoxication are specific for the individual environmental pathogen. Defining these initial pathways will help us to develop specific biomarkers and, hopefully, effective therapies. Here the investigators hypothesize that oxidation of thiols in proteins is an early event in pesticide intoxication and that individual pesticides will specifically affect different proteins, depending on their site of action. The specific aims are designed to identify those proteins that get oxidized following the administration of two different pesticides, rotenone or paraquat, and to validate their potential use as biomarkers in antioxidant therapy. The methods are based on innovative mass spectrometry and fluorescent confocal microscopy techniques.
The candidate is a postdoctoral fellow in the Pittsburgh Institute for Neurodegenerative Diseases (PIND), University of Pittsburgh, under the supervision of Dr. Tim Greenamyre. He received his Ph.D. at the University of Rome, Italy, developing a strong background in biochemistry, molecular biology and mass spectrometry applied to neurodegenerative disorders. In 2003, the candidate moved to the U.S. to join Greenamyre's laboratory and facilitate his career development in an outstanding scientific environment.
The long-term goal of the candidate is to become an established leader in the field of oxidative stress and neurodegenerative disorders, and to provide substantive scientific and social contributions to issues related to public health. The K99/R00 application provides the training requirements necessary to achieve the candidate's ambitions, improving his technical skills, complementing his basic research education with crucial clinical correlations and initiating his career as independent researcher.
The University of Pittsburgh constitutes the ideal environment to develop as an independent scientist, and it ranks among the top in the nation for NIH research support. University core facilities provide state-of-the-art instrumentation and expertise, and the PIND, where the candidate will be based, is designed to be a very collaborative structure, encouraging interactions between established scientists and excellent clinicians.
Exposure to very low doses of pesticides for long periods can generate disorders of the nervous system. This form of intoxication represents an under-appreciated threat to public health, in part because clinical manifestations appear long after the exposure. At that point, when medical diagnosis is possible, neuronal damage is already at advanced stages. The goal of this proposal is to find biomarkers to monitor the progression of intoxication before symptoms appear, in order to develop therapies that can be administrated at earlier stages.
DESCRIPTION (provided by applicant): The lung is constantly exposed to inhaled toxicants that damage the epithelial cells of the conducting airway. Acute injuries involving depletion of progenitor cells are repaired through activation of latent tissue stem cells. However, most environmental injuries are repetitive and result in continual cycles of injury and repair. It is unknown how airway stem cells contribute to repairing repetitive injuries. The hypothesis of this project is that airway stem cells respond to multiple cycles of injury by both proliferating and self-renewing. In injuries accompanied by inflammation, the response of the stem cell hierarchy is dysregulated. To test this hypothesis, nuclear label retention will be used to follow the behavior of airway stem cells in a model of repetitive injury (progenitor cell depletion) that occurs with little inflammation (Aim i). The second and third aims will investigate the hypothesis that injury associated with sustained inflammation impacts the reparative capacity of cells within the bronchiolar stem cell hierarchy. To study the effects of inflammation on lung progenitor cells, a mouse model of silicosis will be used. Silicosis is a fibrotic lung disease caused by long-term inhalation of crystalline silica. Silica exposure rapidly causes inflammatory cell recruitment to the terminal bronchioles and alveolar ducts, regions where stem cells are thought to reside. Impairment of the proliferate responses of the stem cells and transit amplifying cells in the presence of silica will be examined in Aim 2. Finally, TNFa receptor knockout mice will be used to dissect the contribution of inflammation to the impaired regenerative ability of the bronchiolar epithelium in silicosis (Aim 3). The eventual goals of these studies are to determine how silicosis impairs the stem cell hierarchy and to identify therapeutic targets that may halt disease progression. Inhaling silica dust causes scarring in the lung that eventually makes it difficult to breathe. This proposal studies how silica dust impairs the ability of stem cells in the lung to heal lung injuries without scarring. The information learned from this research may help identify new treatments for chronic lung disease. Number pages consecutively at the bottom throughout Form Page 2 the application. Do not use suffixes such as 2a, 2b.
DESCRIPTION (provided by applicant)
The overall goal of the University of Pittsburgh Short-Term Educational Experience for Research (PITT-STEER) in the Environmental Health Sciences for High School and Undergraduate Students is to encourage entry of students into careers in biomedical research and environmental health sciences. The PITT-STEER will exploit a mentored focus on respiratory biology, metal toxicology, health disparities and other contemporary aspects of environmental health science. During the 10-week program, students will conduct original research in these areas under the mentored supervision of Environmental and Occupational Health (EOH) and Behavioral Community Health Sciences (BCHS) faculty in the Graduate School Public Health (GSPH). Students will work on problems at the frontier of biomedical science using state-of-the art techniques. Students will also follow a dedicated series of lectures and participate in problem based learning to introduce basic fundamentals of environmental health science and toxicology. Additionally, they will participate in a seminar program designed to further their understanding of the scientific process and critical analysis, and workshop forums on career paths within biomedical research. The long-term goal is to promote scientific training of new investigators that exploits interdisciplinary approaches to address significant research problems in environmental health science. Prospective students will be selected from a large and outstanding pool of candidates of emerging junior and senior high school students in the Pittsburgh Public School system. Access to this group of students will be facilitated by ongoing efforts in a separate research project (Healthy Class of 2010) in which the Center of Minority Health in the GSPH has established firm interactive ties with the 9 public high schools in Pittsburgh. Six students per summer will be selected by an administrative committee based on high school transcripts, personal statement and letters of recommendation. The Pittsburgh Public School system has significant underrepresented minority (70%) and disadvantaged youth representation facilitating goals of diversity recruitment. Opportunities will be provided to participate in a second summer experience for outstanding students during their own college undergraduate experience. A core of NIEHS and NIH-HLBI funded, experienced investigators has been chosen as mentors. Ancillary activities will include laboratory safety and scientific ethics. At the end of the program, each student will present the results of his/her work at a colloquium of participating students and mentors. A certificate will be awarded to all students successfully completing the program. Longitudinal follow-up will monitor graduate training and career choices elected by student participants in this program and will be compared with that of entering underrepresented minority biomedical graduate students who did not participate in the PITT-STEER or similar programs. This tracking will provide an assessment of the ability of the program to achieve its goals.
DESCRIPTION (provided by applicant): Project Summary: Head and neck squamous cell carcinoma (HNSCC) is thought to be caused by the effects of environmental carcinogens including tobacco, alcohol, betel quid, organic solvents, coal dust, welding fumes, asbestos, and polycyclic aromatic hydrocarbons. The morbidity and mortality associated with HNSCC largely stem from its invasiveness and the high incidence of second primary tumors (SPTs), which are thought to result from "field carcinogenesis," molecular changes in the histologically normal tissue surrounding the primary tumor. Chemoprevention, aimed at counteracting the effects of carcinogens and/or preventing HNSCC SPTs and recurrence, is an important goal. We hypothesize that guggulsterone, a plant- derived compound, may be efficacious as a preventive therapy for HNSCC. Guggulsterone, known for its cholesterol-lowering activity, is available in clinical formulations as a dietary supplement. It has been shown to having anticancer activity in prostate cancer, acute myeloid leukemia, lung cancer and breast cancer cells and, additionally, to suppress the activity of nuclear factor KB (NFKB), a transcription factor that promotes tumorigenesis in HNSCC and other cancers. Our preliminary studies show that treatment of human HNSCC cells with guggulsterone results in growth inhibition, apoptosis, cell cycle arrest and decreases in total and phosphotyrosine (activated) Signal Transducer and Activator of Transcription (STAT)-3, an oncogenic transcription factor known to be constitutively active in most HNSCC tumors. This study will explore the molecular mechanisms of guggulsterone's anticancer activity through in vitro investigation of changes in levels of cell-cycle-promoting and anti-apoptotic genes that are also targets for STATS and NFKB. The mechanism behind the guggulsterone-induced decrease in STATS levels will also be explored. Finally, guggulsterone's chemopreventive effects will be investigated in vivo through the use of two mouse models of HNSCC, a xenograft model and an inducible TGF(3 receptor II deletion/K-Ras mutant, which develops HNSCC tumors similar to those seen in humans. The goal of this work will be to ascertain whether or not guggulsterone may be used for Chemoprevention of HNSCC and to elucidate the molecular mechanism behind guggulsterone's anticancer activity, thus providing a firm basis for its future use in clinical trials. Relevance: HNSCC is an extraordinarily devastating disease and the sixth most common cancer worldwide. Because it is a disease caused by environmental exposures and because high levels of HNSCC- associated morbidity and mortality result from second primary tumors and recurrence, methods of preventing HNSCC have the potential to make a great impact on public health.
DESCRIPTION (provided by applicant): Asbestosis is a debilitating form of pneumoconiosis and interstitial lung disease caused by the inhalation of asbestos fibers. Asbestosis results in fibrotic pathology, which causes significant morbidity and mortality. Inflammation and oxidative stress are known to contribute to the pathogenesis of this disease. Extracellular superoxide dismutase (EC-SOD) is antioxidant enzyme highly expressed in the lung and has been shown to protect the lung from oxidant-mediated damage, inflammation, and interstitial fibrosis. However, the mechanisms through which EC-SOD inhibits pulmonary fibrosis and inflammation remain unclear. Extracellular matrix (ECM) components, such as collagen and glycosaminoglycans, are highly sensitive to oxidative fragmentation and become potent chemoattractants for inflammatory cells. EC-SOD is known to tightly bind and localize to the glycosoaminoglycan, heparan sulfate (HS). The hypothesis of this proposal is that one mechanism in which EC-SOD protects the lung from oxidant-induced damage, inflammation, and fibrosis is by preventing oxidative fragmentation of Heparin/Heparan Sulfate (HS) in the ECM. The proposed studies will utilize in vitro heparin/HS fragmentation assays to study the protective role of EC-SOD and chemotaxis assays to analyze inflammatory responses to oxidatively fragmented heparin/HS and EC- SOD. Finally, wild type mice, EC-SOD knockout and EC-SOD over-expressing transgenic mice will be used to study the in vivo role of EC-SOD and HS in the lungs of asbestos- versus control-treated mice through biochemical and histological analysis.
DESCRIPTION (provided by applicant): Pneumocystis carinii (PC) pneumonia is an opportunistic infection found among HIV-infected patients worldwide. Most infants develop PC-specific antibodies by two years of age, underscoring the frequency at which PC is encountered environmentally. Immuno-intact murine hosts mount protective immune responses leading to subclinical infection, while SCID, CD4 T cell deficient, or B cell deficient mice succumb to PC pneumonia. Yet, mice challenged with PC and thereafter depleted of CD4 T cells resist pneumonia, correlating with high titers of PC specific antibody. While CD4+ T cells appear to be critical in the generation of protective antibodies against PC, maintenance of protective antibodies by memory B cells has not been explored. Further, the specific targets of PC-immunoprotective antibodies have not been fully characterized, particularly their affinity for carbohydrate epitopes, which have recently been shown to be critical in vaccine-based resistance against other opportunistic fungal pathogens. The goal of our efforts is to evaluate the mechanisms whereby antibody based immunity may prevent PC infection, and we approach our studies through two aims. Specific Aim I will evaluate the generation of humoral-mediated immunologic memory against PC through characterization of the kinetics of antibody recall responses in the presence or absence of CD4+ T cells. We will also explore the potential of memory B cells to activate in a CD4+ independent environment and reconstitute PC immunity in SCID mice. Specific Aim II will evaluate whether PC infection elicits specific antibodies against the major surface carbohydrates beta-glucan, mannose, and chitin, and whether memory antibody responses are manifested against these epitopes. Given the affinity of the pattern recognition receptor dectin-1 for PC beta-glucan, we will evaluate whether an antibody-like immunotherapeutic consisting of the extracellular domain of dectin-1 fused to the murine lgG1 Fc fragment enhances PC clearance in SCID mice. Greater understanding of the determinants underlying Pneumocystis-specific humoral immunity and its maintenance will inform on resistance mechanisms against this environmental pathogen and will allow us to optimally vaccinate against PC pneumonia in immunodeficient hosts. PC is a commonly encountered environmental fungus causing lethal pneumonia in HIV+ patients, and little is understood as to how antibodies may prevent infection. Our studies will evaluate the molecules that PC antibodies recognize, and the manner whereby antibody-producing cells remember previous PC exposures, providing insight into normal host resistance and the requirements for vaccine design in the setting of HIV.
DESCRIPTION (provided by applicant): DNA is constantly being damaged by a variety of cellular and environmental agents. In order to prevent DNA mutagenesis, cells have evolved a variety of effective repair mechanisms to correct damaged DNA. However, during replication, not all DNA damage is repaired, rather, damage instead is tolerated by a group of translesion DNA polymerases such as Polymerase eta and Polymerase zeta (Pol C). Evidence in yeast indicates that the catalytic subunit of Pol delta, Rev3, is capable of extending from misincorporated nucleotide templates and is responsible for both damage-induced and spontaneous mutagenesis. Very little is known about the enzyme in mammalian cells. Data suggests that loss of vertebrate Rev3 leads to increased genomic instability and greater susceptibility to the effects of DNA damage. We plan to study the contribution of Rev3 in damage tolerance when wild-type and Rev3 -/- cells are treated with various DNA damaging agents. Furthermore, we will attempt to determine if the polymerase activity is important in damage bypass and whether truncated portions of the protein is sufficient to restore damage tolerance. Finally, data indicates that Rev3 mRNA is regulated at various levels. Mammalian Rev3 protein expression patterns are unknown. We plan to generate an antibody in order to study tissue specific protein expression and whether Rev3 is upregulated during periods of genotoxic stress via immunoblot analysis.
DESCRIPTION (provided by applicant): The overall objective of the proposed project is to gain novel information regarding how components of particulate matter (PM) air pollution and microbial stimuli synergistically interact to promote inflammatory-like processes in the lung, and thereby contribute to the onset of respiratory disease. Numerous epidemiological studies have linked exposure to PM with chronic respiratory and cardiovascular diseases. Studies have shown nickel, a common component of PM, mediates some of the biological effects of PM both in vitro and in vivo. Chronic inflammatory diseases are also associated with colonization by infectious agents, including Mycoplasma fermentans. One of the hallmarks of inflammation is increased elaboration of prostaglandins (PG) through the induction of the cyclooxygenase-2 enzyme (prostaglandin-endoperoxidase synthase 2, PTGS2). Preliminary data show that exposure to NiS04 and the M. fermentans-derived lipopeptide MALP-2 synergistically enhances expression of PTGS2 and the immune-modulating chemokine CXCL8(IL-8) in cultured human lung fibroblast cells (HLF). Concurrent with the induction of PTGS2 and CXCL8, Ni and MALP-2 synergistically stimulate PGE2 and CXCL8 protein release from HLF. The proposed study will explore the hypothesis that PTGS2-derived PGE2 contributes to the amplification of CXCL8 production from HLF during mixed exposures to Ni and microbial stimuli. The specfic aims are to 1) determine the cellular and molecular mechanisms underlying the synergistic induction of PTGS2 expression in HLF in response to concurrent Ni and MALP-2 exposure and 2) determine the mechanistic relationship between the production of PGE2 following mixed exposures to Ni and MALP-2 and subsequent production of CXCL8. Luciferase reporter plasmids driven by human PTGS2 and CXCL8 promoters along with electrophoretic mobility shift assays (EMSA) will be employed to determine how Ni and MALP-2 synergistically enhance activation of PTGS2 and CXCL8. Relevant cis-regulatory DNA binding elements and transactivating protein factors responsible will be mapped using full-length, truncated and mutant versions of the PTGS2 and CXCL8 promoters. siRNA along with pharmacological agonists and antagonists will be used to probe the specific contribution of PTGS2, PGE2 and PGE2 receptor subtypes involved in transducing Ni and MALP-2-induced CXCL8 expression and subsequent protein release from HLF. Real-life environmental exposures are likely to involve a mixture of toxicants and microbial stimuli. Results from this study will increase our understanding of how metals such as Ni can sensitize cells to microbial-driven inflammation in the lung, and how toxicant and microbial stimuli interact to promote the onset of respiratory disease in human populations.
DESCRIPTION (provided by applicant): Pneumoconioses are occupational lung diseases caused by the inhalation of dust fibers/particles such as asbestos and silica. Experimental models of asbestosis recapitulate the salient features of idiopathic pulmonary fibrosis (IPF), a debilitating disease with both high morbidity and mortality due to a lack of effective therapies. Recent epidemiological studies have suggested that diabetics are at increased risk of developing idiopathic pulmonary fibrosis. This has led our lab to hypothesize that diabetics are at increased risk of developing pulmonary disease after environmental exposures. As the diabetic population increases at an alarming rate it is important to determine the mechanism by which diabetics are more susceptible to pulmonary injury so that it can be better prevented and treated. Recently our lab has implicated loss of the receptor for advanced glycation end-products (RAGE) as a key pathogenic step in the development of pulmonary fibrosis. This receptor has also been implicated in numerous other diabetic pathologies including neuropathy, atherosclerosis, and nephropathy. This has led us to investigate its potential role in increasing diabetics susceptibility to pulmonary injury. Preliminary studies have shown that aged diabetic mice have a loss of RAGE in their lung tissue. This could provide a potential mechanism by which diabetics are more likely to develop pulmonary fibrosis. This project will focus on the mechanisms by which RAGE promotes wound healing and prevents de-epithelialization and subsequent disease. We will also investigate the role that increased advanced glycation end-products (AGEs) play in the disease pathogenesis. Ultimately, we hope to determine if clearing RAGE ligands by use of a non-signaling decoy receptor, sRAGE, protects against the fibrotic response after asbestos exposure. This project will investigate the possibility that diabetics are at increased risk of developing pulmonary fibrosis after being exposed to occupational dust particles. It will provide a better understanding of how the disease develops which will help us develop better ways to prevent and treat the disease and its symptoms.
DESCRIPTION (provided by applicant) This multidisciplinary program supports 3 predoctoral and 1 postdoctoral fellows per year at Thomas Jefferson University (TJU) for training in birth defects research. Its goal is to provide the basic and applied skills necessary to become independent investigators in research to uncover genetic and molecular control of cellular function in developing systems and to apply these principles to understanding mechanisms of developmental defects caused by environmental toxicants. One in 33 babies is born with a serious structural malformation. Two-thirds of these cases have no obvious cause. Because of human health concerns about the developmental toxicity of environmental agents, scientists and regulators have focused efforts on understanding and protecting against potential hazards of these agents to developing embryos, fetuses, and children. TJU historically comprises one of the world's largest academic centers for birth defects research and has contributed substantially to the pipeline of investigators in this important research area. The Program reflects a modern understanding that developmental defects are best studied through gene-environment interaction and with newer technologies for high-throughput measurement and functional analysis. Resources come from a core of well-funded faculty in several departments at Jefferson including the new Daniel Baugh Institute for Functional Genomics and Computational Biology. Research interests of these faculty fall into three overlapping categories: 1) genetic signals and responses during normal embryonic development; 2) mechanisms of developmental toxicity and teratogenesis; and 3) functional genomics and computational biology. Applied skills are provided by opportunities for formal collaboration with scientists at major pharmaceutical companies and teratology contract laboratories in the Philadelphia-New Jersey region. Program integration is enhanced by a broad core curriculum in cellular, molecular, and developmental biology with focused training in developmental toxicology, teratology, and biostatistics. Students are required to attend and participate in weekly seminar and journal club programs. Duration of training averages 5-years for predoctoral students and 3 years for postdoctoral fellows, with 2 years of training grant support per individual. The Program has a successful track record of minority trainees.
DESCRIPTION (provided by applicant): Socioeconomic status (SES) during childhood and adolescence is an important predictor of adult health. Even so, there is little known about how childhood SES (chSES) influences long-term health trajectories. Here, the proposed project tests the hypothesis that chSES affects the development of key physiological systems (autonomic nervous system [AIMS], hypothalamic-pituitary adrenal cortical [HPA] axis, immune system) that in turn may influence risk for morbidity and mortality as an adult. The researchers place a special emphasis on identifying the environmental exposures and accompanying psychological mechanisms by which chSES may influence the developmental trajectory of these physiological systems. The researchers assess SES, social, physical and family environments during childhood, adolescence and adulthood by interview and then measure the stress-reactivity of the above mentioned physiological systems during adulthood. In regard to the specific aims, the researchers will first develop a uniform and comprehensive assessment of childhood SES and childhood environments. Within a stress-reactivity paradigm, the researchers hypothesize that adults who experienced lower chSES will possess more negative interpretations of ambiguous behavioral stressors and increased stress-evoked cardiovascular, neuroendocrine, and immune reactivity. This effect may be mediated by differential exposures to detrimental physical, social, and family environments during childhood. These environments may influence the development of psychological traits/states, behavioral health practices, and physiological systems with implications for adult health. Overall, the study and training supported by the F30 NRSA will attempt to identify the underlying physiological mechanisms through which chSES influences adult morbidity and mortality. A novel emphasis is placed on identifying characteristics of the childhood physical, social, and family environments that may mediate the hypothesized relation between chSES and stress related changes in the ANS, HPA axis, and immune system.
DESCRIPTION (provided by applicant)
The interest in and need for increased environmental health research has steadily grown during the past decade. To address this need, the National Institute of Environmental Health Sciences (NIEHS) has established goals for increased numbers of tomorrow's environmental health scientists. To this end, the focus of the Short Term Educational Experience for Research summer program at the University of Pennsylvania will help to expose selected undergraduate and high school students to the field of environmental health science through mentorship, coursework, and field experience. This program represents a collaboration of faculty and staff from the Center of Excellence in Environmental Toxicology, a cross disciplinary research initiative, the Penn Summer Mentorship Program for High School Students sponsored by the Office of the Provost, and faculty from the School of Medicine, Nursing, and Arts & Sciences. The program will provide didactic experiences in environmental and public health and provide mentorship opportunities in the defined areas of environmental exposures and health effects associated with lung and airway disease, endocrine and reproduction disruption, gene-environment interactions and oxidative stress.
DESCRIPTION (provided by applicant): In this proposed research, benzo[a]pyrene (B[a]P) and it's metabolites, will be used as model compounds to determine the major metabolic pathways of PAHs in lung carcinogenesis. It is generally accepted that cytochrome P4501A1, CYP1A1, is primarily responsible for the metabolism of B[a]P to 7,8-dihydroxy-9,10- epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (B[a]PDE), the ultimate DNA-adduct forming, carcinogenic reactive metabolite; thus connecting CYP1A1 to the toxicological effects of this environmental carcinogen. DNA-adduct formation has been detected at CpG islands located in the promoter region of the tumor suppressor p53. Inactivation of p53 by point mutations is seen in over 40% of all cancers. However, in preliminary research using H358, human bronchoalveolar lung cells, it was found that DNA-adduct levels were higher in non-treated CYP1A1/1B1 null cells compared to H358 cells in which CYP1A1 expression was induced with 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD). This data correlates with literature results of CYP1A1 KO mice stuides in which it was determined that CYP1A1 conferred a protective rather than a toxic effect. This is indicative of the metabolic activity of another CYP in the metabolism of B[a]P. Several other CYP enzymes are capable of catalyzing this metabolism. Preliminary data has also indicated that TCDD may have an effect on the transport of B[a]P-reactive metabolites. It is known that TCDD is able to induce several of the ABCC drug transporter family members; therefore, explaining why less DNA-adduct formation is observed in lung cells pretreated with TCDD . This proposal, which has two aims, will identify the predominate CYP responsible for B[a]P metabolism in the lung and determine the role of B[a]PDE transporters in DNA-adduct formation in lung cells. A stable isotope dilution LC-MRM/MS method, previously developed, will be used for DNA-adduct quantification. DNA-adduct levels will be correlated to mRNA and protein expression determined by RT-PCR and a novel quantitative proteomics method, SI LAC. CYPs including 1A1, 1B1, 1A2, 2B6, 2E1, 2J2, 3A4, and 3A5 will be investigated. DNA-adduct formation will also be monitored in lung cells in which drug transporter expression has been inhibited by siRNA. Continuing research in this area will provide exciting new therapeutic strategies (drug targets) that can be employed to prevent PAH-mediated DNA damage. Relevance to public health: Cigarette smoke is known to cause 90% of lung cancer, but only 10% of smokers develop lung cancer. Examination of alternative CYP metabolism and B[a]PDE transport in lung cells will provide an experimental model to explore whether inter-individual differences in CYP metabolism and transport contribute to these phenomenon.
DESCRIPTION (provided by applicant): Immunologically mediated diseases such as asthma, allergies, and autoimmunity are important causes of morbidity. Naturally occurring regulatory T cells (NatTregs) show significant promise as potential tools/targets for immunomodulatory therapies. NatTregs require interleukin (IL) 2 and T cell receptor (TCR) activation to gain maximal suppressive ability. Regulatory T cells can be induced (IndTregs) de novo from T helper (Th) cells by activation in the presence of TGFbeta. However, little is known about the influence of pro- inflammatory cytokines on Treg function and phenotype. Recent studies including our own suggest an important regulatory role for the proximally induced pro-inflammatory cytokine, IL-6, and the cytokines critical for the adaptive immune response, IL-4 and IFN-gamma, in the development and function of NatTregs and/or IndTregs. The central hypothesis that will be developed in this proposal is that the generation and function of Tregs are influenced by specific cytokines. This has important ramifications in use of Tregs in therapy. To test this hypothesis, we will: Aim 1: Investigate the effect of IL-6 on FoxPS expression and suppressive function in NatTregs. Aim 2: Characterize modulation of IndTregs by cytokines during and after induction. A model of Aspergillus hypersensitivity will be used to investigate IndTreg function in vivo.
DESCRIPTION (provided by applicant)
The Tony Mazzocchi Center (TMC), formed by the Paper, Allied-Industrial, Chemical, and Energy Workers International Union (PACE) and the Labor Institute, proposes a model program to train workers at DOE facilities to reduce exposures to hazardous materials, waste operations and incidents. TMC training will consist of 1,714 classes for 40,000 DOE employees who will receive 304,000 hours of training over the 5-year grant period. This training, which conforms to OSHA Standard, 29 CFR 1910.120 (sections p and q), will help workers to protect themselves during emergencies and to implement strategies to prevent potentially deadly accidents. The courses include, a) 40-hour General Site Worker Training classes for Hazardous Waste Site workers; b) 16-hour Incident Investigation Training for labor and management teams and awareness classes for TSD (Treatment, Storage, Disposal) site workers; c) 16-hour Labor-Management Committee Training on Site Emergency Response Plans and Near-miss programs; d) 8-hour Annual Refresher Training required for all site workers to maintain their certification; e) 8-hour Systems of Safety courses at TMC Integrated Training Sites-Integrated Safety Management Systems; f) 40-hour Lessons Learned classes for site worker-management teams; and g) 8- and 12-hour Lessons Learned/Near Miss Identification refresher courses.
Specific Aims are to, 1) develop 44 worker-trainers through annual 40-hour technical training workshops and quarterly site workshops; 2) expand partnerships with DOE site contractors, regulatory personnel and DOE training managers; 3) continue Advanced Training Technology Initiatives (ATTI) that include web-based Lessons Learned training and e-learning; 4) promote a culture of continuous learning by developing a Lessons Learned Initiative that will integrate the TMC ongoing lessons learned program with the DOE's lessons learned system; 5) expand model team-based research and evaluation; 6) expand Integrated Training Site programs that connect training to Integrated Safety Management Systems (ISMS); 7) pilot a new Near-miss Initiative program with the Wharton Risk Management and Decision Processes Center of the University of Pennsylvania; and 8) participate fully in the Multigrantee Trainer, Curriculum, and Evaluation Initiative that brings together seven unions to encourage and disseminate innovation through collaboration on training, curricula and evaluation. The TMC also will form a nationally integrated network of support that includes, the University of Massachusetts (UMass) at Lowell for technical assistance and advanced training technologies; the TMC Advisory Board; the Labor Institute for support in developing trainers and producing materials and the New Perspective Consulting Group, Inc. for evaluation.
DESCRIPTION (provided by applicant)
The Tony Mazzocchi Center (TMC) formed by The Paper, Allied-Industrial, Chemical and Energy Workers International Union (PACE), the United Steelworkers of America (USWA) and the Labor Institute (together representing over 875,000 industrial workers) will conduct over the next 5 years approximately 3,800 workshops reaching 86,000 workers and community residents with 900,000 hours of training to prevent hazardous substance emergencies and to protect workers and communities should such accidents occur. The model training will consist of courses that meet the requirements of 29 CFR 1910.120, address environmental justice and health disparities, and assist worker-trainer development and evaluation. Specific Aims include to, 1) expand model site-based training, called ITS programs, to meld 1910.120-based training into broad, ongoing plant-wide prevention programs. The TMC will add 8 ITS programs per year (total of 40 new sites) to our existing 35 sites resulting in 72 total sites at the end of five years involving an estimated 30,000 workers and managers. In addition, the goals are to, 2) pilot a new Near-miss Initiative working with the Wharton Risk Management and Decision Processes Center of the University of Pennsylvania; 3) conduct community/labor workshops with regional environmental justice networks and Latino workers; 4) expand the use of advanced training technologies including web-based lessons learned, e-learning and emergency response simulation programs; 5) use participatory research and evaluation to monitor and strengthen the program; and 6) lead a multi-grantee (seven unions) initiative to work together on trainer, curriculum and evaluation development. With the HDPTP supplement the TMC will help protect workers and community residents from potential exposures to hazardous materials released during terrorist attacks on industrial facilities with large volumes of highly hazardous chemicals, specifically at the 306 Risk Management Program Sites (RMPs) at which PACE and USWA represent 75,000 workers. The TMC will reach 19,700 participants with 192,360 contact hours of weapons of mass destruction (WMD) prevention, preparedness and response training. The TMC will also recruit and train an entirely new, nation-wide, industrial catastrophic incident Prevention, Preparedness and Response (PPR) Corps composed of 68 experienced workers from RMP sites. The PPR Corps, consisting of credentialed industrial responders, will work with OSHA, and through OSHA, the DHS to assist in OSHA's Regional Emergency Response Plans for high hazard sites. The PPR Corps will train industrial site workers, management and community responders, as well as conduct facility audits to evaluate prevention activities, emergency preparedness, and readiness to create site safety plans in the event of a catastrophe.
DESCRIPTION (provided by applicant)
Reducing the population prevalence of obesity is a current major public health goal. Interventions to reduce the prevalence of obesity have generally focused on individual behavior and lifestyle but have met with limited success. Strategies that focus on the role of the built environment have been neglected. The purpose of this innovative pilot study is to evaluate, using a quasi-experimental design, the impact on diet and psychological health of a three-year $40 million state-government funded program - The Pennsylvania Fresh Food Financing Initiative - that aims to improve the local built food retail environment in Philadelphia. The project has four specific aims. 1) To describe and compare fruit and vegetable consumption patterns and measures of psychological health in an intervention neighborhood against a matched comparison neighborhood. 2) To evaluate whether these patterns change after the opening of a new food supermarket (the intervention) in the intervention neighborhood compared to a matched comparison site. 3) To explore impacts on defined subgroups of residents based on income, education and baseline consumption status. 4) To investigate changes in the retail economy in the intervention neighborhood and compare these with the comparison neighborhood. A telephone survey of residents of two Philadelphia neighborhoods (one intervention and one comparison) with an achieved sample size of four hundred and sixty-six men and women aged 18+ in each neighborhood at follow-up will be undertaken. At baseline, respondents will be contacted with a pre-notification letter which will then be followed by a telephone call designed to elicit responses to questions relating to diet, mental health, perceptions of food access, food shopping behavior, transport and a range of socio-demographic data. Respondents will then be followed-up at eight months in order to assess the effect of the intervention. In addition geographical information systems will be used to assess positive and negative changes in the local food retail economy and relate them to changes in physical access to food. Findings from the project will be used to prepare a proposal to NIH for a larger mixed-method, multi-site experimental study in a range of community settings (urban, small town, rural) throughout the USA.
Crisp Terms/Key Words: experimental design, outcomes research, behavioral /social science research tag, clinical research, socioenvironment, socioeconomics, health behavior, health survey, longitudinal human study, vegetable, fruit, nutrition related tag, dietary constituent, diet, nutrient intake activity, mental health, statistics /biometry, questionnaire, interview, human subject, public health, food resource, food chain, disease /disorder proneness /risk, obesity, body weight
DESCRIPTION (provided by applicant): A multi-scale strategy is proposed to develop, couple, apply, and validate multimodality imaging and physics modeling of resolvable and sub-resolvable scales in human respiration. High-resolution computed tomography (HRCT) will be used to characterize the "macroscale" convective range of the lung. Microscopic computed tomography (.CT), and confocal microscopy (CLSM), will be used to characterize the "microscale" global and cellular architectures of the respiratory units. Multiphase computational fluid dynamics and quasi-one-dimensional functional modeling will be used to simulate the multi-component fluid mechanics at the macro- and micro-scales, respectively. Software infrastructure and two-phase fluid mechanics models will be developed to address the coupling between the physics at these two scales. Model predictions will be validated against experimental and clinical data from the literature. A novel and critical element of the proposed research is that the interfaces between functional biological scales will be developed using recent dimension-reducing coupling strategies developed in the nuclear reactor safety/simulation community, and multidisciplinary data-exchange standards developed in the aerospace sciences community. Coupling technologies will be developed between macro- and microscales, and between imaging and physical modeling; these will yield a system-level model that accommodates the critical two-way coupling between convective respiration physics and uptake, deposition, and disease-state morphology. Such an integrated approach will elucidate heretofore inaccessible physical understanding, dependencies, and treatment implications. The coupling software to be developed will be modular and open-source so other investigators can "plug-in" their models at the macro- and micro-scales, and/or evolve the system to other organs or human systems such as the liver or kidney. The ultimate public health goal of the research is improved understanding of respiratory function and disease, and evaluation/assessments of the effects of therapies, injury, surgical intervention, and aging on lung structure and function. The physics-based coupling between multiple scales is a critical step towards a complete integrated physiological model of the human respiratory system: a "virtual human lung."
DESCRIPTION (provided by applicant):
Exposure and biological response biomarkers of cigarette smoke. Exposure to tobacco smoke (mainstream and environmental) is a leading cause of death in the US. Cigarette smoke is an extremely complex mixture, which some 3800 constituents including numerous polycyclic aromatic hydrocarbons (PAHs), in both the mainstream and sidestream (environmental) smoke fractions. Cigarette smokers provide an extreme model of PAH exposure that will permit both exposure and biological response biomarkers to be developed. There is substantial evidence that PAHs are causative agents in lung, skin, and bladder cancer. Furthermore, tobacco smoke is associated with oxidative stress, pancreatic cancer, cardiovascular disease, and chronic obstructive pulmonary disease (COPD), although the specific role of PAHs is not clear. Interestingly, the cardiovascular effects of sidestream smoke are almost as great as mainstream smoke. The present proposal stems from significant advances we have made over the last six years in the quantification of protein, lipid, and DNA biomarkers using stable isotope methodology and our basic research into enzyme regulation during oxidative stress. Previous methods for analyzing oxidative DNA damage have been fraught with numerous methodological problems so that the current state-of-the-art involves the use of a COMET assay to measure 8-oxo-2'-deoxyguanosine (dGuo) lesions. We have recently devised a more quantitative method based on immunoaffinity stable isotope dilution liquid chromatography- tandem mass spectrometry (LC-MS/MS) that can be readily elaborated to studies of tobacco smokers. We also showed that oxidative stress could induce the formation of aldo-keto reductases (AKRs) of the 1C family. AKR1C3 is the enzyme, which we recently showed is responsible for the conversion of prostaglandin (PG) D2 to the potent bronchoconstrictor 11p-PGF2. This provides an additional potential link between oxidative stress and COPD as well as the potential for a new therapeutic strategy, which involves AKR1C3 inhibition. Finally, preliminary studies have revealed that a DNA-adduct than can only arise from lipid peroxidation is present in the urine of cigarette smokers but is completely absent in urine from non-smokers. We propose to build on these exciting new findings by developing panels of in vivo biomarkers of exposure and biological response, which we hypothesize will make it possible to distinguish a cohort of non-smokers from a cohort of disease-free tobacco smokers. The hypothesis will be tested by conducting research under the following three specific aims: Aim 1. To discover whether B[a]P and B[a]P-7,8-dione induce AKR1C/2 in NHBE cells and increase oxidative stress to form 8-oxo-dGuo and HedGuo in DNA, induce AKR1C3 in HASM cells and increase the biosynthesis of the potent bronchoconstrictor 11p-PGF2, as potential urine and EEC biological response biomarkers of PAH exposure. Aim 2: To discover secreted proteins following treatment of NHBE and HASM cells with B[a]P and its oxidative metabolites as potential serum biological response biomarkers of PAH exposure. Aim 3: To conduct predictive and refinement analyses of in vivo exposure and response biomarkers in urine together with biological response biomarkers in EBC and serum in order to distinguish non-smokers from disease-free tobacco smokers. Successful completion of the proposed research will provide a panel of biomarkers of exposure and biological response to tobacco smoke will have significant utility in future studies designed to elucidate the relationship between gene environment interactions and diseases such as cancer, cardiovascular disease, and COPD.
DESCRIPTION (provided by applicant): Even without signs of external injury, chemical exposure can produce severe trauma to internal target organs including the lungs, heart, gastrointestinal tract, eyes, and the central nervous system. Of these injuries, the extent of lung injury often is the most critical to survival. Chemical Induced Acute Lung Injury (CIALI) can be viewed as a molecular cascade mounting over hours and days subsequent to even a transient incident. Unfortunately, CIALI is a likely consequence of terrorist attacks of multiple possible scenarios including intentional detonation of chemical plants, railroad car derailment, or chemical truck hijacking. Chemicals of high concern include chlorine, phosgene, sulfuric acid, ammonia, and acrolein. Predictive strategies will require the monitoring of multiple biochemical indicators forming complex molecular signatures. Our goal is to understand the genetic, global transcriptomal, and molecular events that will provide insights into the mechanisms of CIALI and could redirect or strengthen current emergency clinical approaches to diagnosis and treatment. The objective of this application is to determine the molecular mechanism(s) and therapeutic efficacy of TGFalpha and FGF7 in enhancing survival in this condition. Our central hypothesis is that the interplay between TGFalpha, FGF7, and TGFbeta signaling determines survival and controls the susceptibility to sequelae from CIALI. To explore our hypothesis, we seek to: 1) Identify the genetic determinants and molecular mechanisms controlling CIALI common to exposure to 5 leading hazardous chemicals: chlorine, phosgene, sulfuric acid, ammonia, and acrolein, 2) Evaluate the therapeutic efficacy of TGFalpha and FGF7 induction and signaling during CIALI and determine whether pulmonary fibrosis is a necessary sequela as a consequence of protection, and 3) Identify the molecular mechanisms that are unique to each of the 5 leading hazardous chemicals during the early development of CIALI. At the completion of this project, we expect to: 1) Identify novel genetic differences that determine the susceptibility to CIALI, 2) Identify the events modulated during CIALI that are common to multiple agents 3) Evaluate the effectiveness of therapies by that lead to protection in CIALI, 4) Determine whether pulmonary fibrosis is an untoward consequence of activating TGFalpha/FGF7signaling during CIALI, 5) Develop an initial chemical specific database on the selective signatures of the 5 leading hazardous chemicals.
DESCRIPTION (provided by applicant)
Environmental exposures contribute to the development and progression of human disease, including cancer, cardiovascular disease and neurodegeneration. Detailed study of the cellular and molecular impact of environmental exposures provides insight into disease etiology as well as identifying humans predisposed to environment related disease. Many environmental exposures induce DNA damage and can impact DNA repair, damage tolerance and/or cellular responses to DNA damage, leading to the accumulation of DNA lesions, mutations, chromosomal aberrations or cell death. Therefore identifying environmental exposures that promote genotoxic stress and understanding how cells respond to DNA damage is critical for understanding cancer and degenerative diseases including aging.
The Annual Midwest DNA Repair Symposium (AMDRS) provides a regular forum for deliberating basic and applied research focused on DNA damage and repair as it relates to human genetic diseases, environmental exposures, cancer and aging. The 10th AMDRS will bring together scientists from the U.S. Midwestern States with the goal of enhancing collaborations and disseminating current knowledge about the mechanisms of DNA repair and their influence on environmentally induced DNA damage and human disease. The basic objectives guiding the conduct of this symposium were established in 1999 at the first meeting in Ann Arbor, MI. The three specific aims of the AMDRS are: (1) to provide a venue for scientists, from principally Midwestern states, to present and discuss the current research in the field; (2) to provide students, postdoctoral fellows and junior independent investigators the opportunity to present their work to peers and interact with leaders in the field, and (3) to enhance interactions between researchers and foster collaborations to address important issues in the field of environmentally induced DNA damage and its impact on human disease.
A congregation of innovative ideas and novel observations and their dissemination in an intimate setting is anticipated to promote meaningful cross talk amongst researchers in the field leading to rational strategies for combating disease. A second important purpose served by AMDRS is to provide a forum for graduate students and postdoctoral fellows to present their work and interact closely with high caliber scientists studying the repair of DNA damage caused by environmental exposures. Topics of discussion will include gene-environment interactions that modulate responses to genotoxic stress, the impact of DNA damage on human cells and developing model systems to study genome instability syndromes. The AMDRS has provided a very economical and effective scientific forum for the past nine years, fostering interactions, forging new collaborations and helping to attract young investigators to careers in environmental health sciences.
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