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Record Count: 54
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DESCRIPTION (provided by applicant): Toxicity of many xenobiotics has been associated with lipid peroxidation (LPO) caused by reactive oxygen species (ROS) generated during their metabolism. Among the antioxidant enzymes, only glutathione peroxidases (GPxs) are known to provide protection against LPO by reducing lipid hydroperoxides. The ?-class glutathione S-transferases (?-GSTs), also show GPx activity towards lipid hydroperoxides, but the physiological role of this activity is not understood. Our preliminary studies show that human hGSTA1-1 and hGSTA2-2 can reduce membrane phospholipid hydroperoxides (PL-OOH) in situ by their GPx activity. Over expression of these enzymes in K562 cells attenuates oxidant (H202 or xenobiotic) induced LPO and cytotoxicity. The over expression of another ?-GST, hGSTA4-4 which detoxifies the end- product of LPO, 4- hydroxynonenal (4-HNE), by conjugating it to glutathione, also protects against oxidant toxicity. We hypothesize that GSTs provide a second line of defense against ROS and act as antioxidant enzymes, which protect cells against toxicity of oxidants/xenobiotics by attenuating LPO. The following specific aims are proposed to test this hypothesis: 1. The physiological significance of the ?-GSTs, GSTA1-1 and GSTA2-2 will be studied by determining their kinetic properties towards LPO products, PL-OOH and 4-HNE. Their contributions in the reduction of PL-OOH in rat and mouse liver will be determined and compared with those of the seleno GPxs. We will examine if the cells over expressing these enzymes are protected against H202 or oxidant xenobiotics (e.g., doxorubucin, CCI4) induced activation of c-Jun N-terminal kinase (JNK), caspase 3, and subsequent apoptosis. 2. Cells will be transfected with the ?-GST isozyme, GSTA4-4, which detoxifies 4-HNE and the toxicity of H202 and oxidant xenobiotics which induce LPO will be compared in the transfected and control cells. Xenobiotics, H202, xanthine/oxidase, DOX induced toxicity and apoptosis will be compared in the control and transfected cells to delineate the role of 4-HNE and GSTA4-4 in oxidative stress mediated signaling. 3. We will examine whether the toxicity of oxidants is enhanced in GSTA4-4 knock out mice because of their inability to detoxify 4-HNE. Since GSTs can be induced by non-toxic micronutrients, these studies will help in devising strategies for negating the toxicity of environmental chemicals and chronic oxidative stress, which leads to age, related degenerative disorders.
Crisp Terms/Key Words: JUN kinase, terminal nick end labeling, oxidative stress, glutathione transferase, cytotoxicity, peroxidation, glutathione peroxidase, free radical oxygen, xanthine oxidase, antioxidant, hepatotoxin, lipid peroxide, immunoprecipitation, immunocytochemistry, histopathology, laboratory rat, laboratory mouse, transfection
DESCRIPTION (provided by applicant): Environmental hazards and stress, carcinogens, and anti-cancer therapeutics cause apoptotic cell death through the intrinsic or mitochondrial death pathway. Abnormal apoptotic response often contributes to tumor development and defects in apoptosis are intimately associated with tumor cell resistance to anti- neoplastic agents. Caspase activation lies in the core of apoptotic cell death. In the mitochondrial death pathway, cytochrome c (CC)-initiated Apaf-1 apoptosome formation represents a key initiating event in caspase-9 (an initiator caspase) activation, which ultimately activates effector caspases such as caspase-3 to execute cell demise. How Apaf-1 apoptosome is formed and regulated in vivo (i.e., in the stimulated cells) remains poorly understood. Recently, we provided evidence that physiological levels of nucleotides inhibit the CC-induced, apoptosome-mediated caspase-9 activation by binding directly to CC and preventing CC from interacting with Apaf-1 (Chandra et al., Cell 125, 1333-1346, 2006; Appendix I). Consequently, the CC- mediated apoptosome assembly and activation are blocked. Co-microinjection of nucleotides and CC renders cells resistant to the CC-induced apoptosis in vivo whereas experimentally reducing nucleotides enhances both CC and apoptotic stimuli-induced cell death. These observations lead us to hypothesize that physiological levels of nucleotides, in addition to their well-established roles in nucleic acid synthesis, intermediate metabolism, and maintenance of bioenergetics, also function as critical prosurvival factors by directly inhibiting the CC-mediated apoptosome formation and caspase activation. There are several critical unanswered questions related to Apaf-1 apoptosome regulation by nucleotides. We propose the following three Specific Aims to address some of these questions: 1) To further study nucleotide interaction with CC in vitro and to elucidate nucleotide interaction with CC in vivo; 2) To investigate nucleotide interaction with Apaf-1 and its impact on apoptosome activation; and 3) To study the effects of Apaf-1 isoform expression and nucleotide interaction on Apaf-1 apoptosome activation. These goals will be achieved by a combination of cell biological, biochemical, and molecular approaches. The accomplishment of the proposed goals will greatly advance our understanding of apoptosome regulation in vivo and help us understand cellular response to environmental stress and cancer cell response to anti- tumor therapeutics.
DESCRIPTION (provided by applicant): Environment hazards and stress, experimental and environmental carcinogens, environmental exposure to UV and ionizing radiation, and anti-cancer therapeutics (including chemotherapeutic drugs, radiation, and adjuvant therapeutics) cause cell death through intrinsic or mitochondrial death pathway. Abnormal apoptotic response contributes to tumor development induced by environmental hazards and carcinogens. Defects in apoptosis are also intimately associated with tumor cell resistance to anti-neoplastic agents. Elucidating the molecular mechanisms of the mitochondrial death pathway will facilitate understanding of how cells respond to environmental stresses, hazards and therapeutics. The essence of apoptosis in various signaling pathways converges on the activation of caspases. Caspase-8 acts as the most upstream caspase in apoptotic signaling initiated by Fas (CD95), TNFR, and TRAIL receptors. Normally, engagement of death receptors on the plasma membrane recruits FADD and procaspase-8/10 to the death-inducing signaling complex (DISC) to activate caspase-8, which ultimately activate caspase-3 to execute apoptosis. Recent evidence, however, indicates the presence of significant levels of caspase-8 on the mitochondria. How mitochondrially-localized caspase-8 is activated and regulated at the mitochondrial levels remains unknown. Our recent published and preliminary observations demonstrate that: 1) a variety of intrinsic apoptotic stimuli activate caspase-8 in epithelial cancer cells and mitochondria appear to function as a critical platform for FADD-mediated caspase-8 activation in these apoptotic systems, and 2) FADD on the mitochondria, upon apoptotic induction, oligomerizes to higher molecular weight protein complex. Accordingly, we hypothesize that mitochondria function as crucial signaling center to initiate FADD-mediated caspase-8 activation and DISC assembly during apoptosis induced by intrinsic stimuli. The initiation of caspase-8 activation on the mitochondria may represent the earliest event or one of the earlier events in parallel with caspase-9 activation during apoptosis induction. Elucidation of the afore-mentioned novel regulatory mechanisms should not only help us better understand how caspase activation is initiated and regulated but also lay a foundation for designing novel anti-cancer therapeutics. Two Specific Aims are proposed to test the hypothesis: 1). To establish that intrinsic apoptotic stimuli induce FADD-mediated caspase-8 activation on the mitochondria; and 2). To characterize the formation and activity of DISC-like complex on the mitochondria and its regulation by antiapoptotic proteins (i.e., Bcl-xL, c-FLIP). Environmental hazards and stress, experimental and environmental carcinogens, environmental exposure to UV and ionizing radiation cause cell death or apoptosis. Elucidating the molecular mechanisms of apoptotic cell death will facilitate the understanding of how cells respond to environmental stresses and hazards, and will be highly significant in improving human health.
DESCRIPTION (provided by applicant): The primary goal of the research proposed in this grant application is to identify and characterize genes that modify susceptibility to skin tumor promotion using the multi-stage skin carcinogenesis model in mice. Much data from both human epidemiologic and animal studies support the hypothesis that cancer susceptibility in the general population is a function of multiple, poorly penetrant modifier genes that control the propensity toward environmental carcinogen-induced tumor development. Data suggest that susceptibility to the tumor promotion stage is a major determinant of overall susceptibility to multi-stage, epidermal carcinogenesis in mice. Identifying and characterizing genes that modify susceptibility to tumor promotion is crucial for a complete understanding of multistage carcinogenesis and for developing effective cancer prevention strategies. Genes that modify susceptibility and severity of other disease syndromes in humans have successfully been identified in animal models using approaches similar to those proposed in this application to identify and characterize promotion susceptibility loci. To identify genes that modify promotion susceptibility, we have used the classic multi-stage skin tumorigenesis model in the mouse, which is an excellent animal model to study epithelial carcinogenesis in humans. Genetic control of susceptibility to skin tumor promotion by the phorbol ester, 12- O-tetradecanoylphorbol-13-acetate (TPA), in crosses between susceptible DBA/2 and resistant C57BL/6 mice is a multigenic trait and we have mapped promotion susceptibility loci to chromosomes (chr) 1 (Psl3), 2 (Psl2), 9 (Psl1), and 19 (Psl4). Analysis of C57BL/6.Psl1dba congenic mouse strains suggests that at least three genes underlie the effects of Psl1 on skin tumor promotion susceptibility. We have designated these loci as Psl1.1, Psl1.2, and Psl1.3. Furthermore, global gene expression analyses using cDNA microarrays revealed that glutathione S-transferase alpha 4 (Gsta4), which maps within Psl1.2, is expressed at 20-fold higher levels in the epidermis of TPA-treated C57BL/6 compared to DBA/2 mice. Gsta4 is a glutathione-S-transferase (GST) and a major substrate for Gsta4 is 4-hydroxy-2(E)-nonenal (4-HNE), a product of lipid peroxidation. Recent preliminary studies indicate that C57BL/6 mice, null for Gsta4, are more sensitive than wild-type mice to the multi-stage skin tumor protocol, using TPA as a promoter. These observations, taken together with published data supporting a role for Gsta4 in response to oxidative stress, suggests that Gsta4 is a good candidate for a gene that underlies the effect of Psl1.2 on TPA promotion susceptibility. In the proposed research, we will test the hypotheses that Gsta4 modifies the response to TPA skin tumor promotion in DBA/2 and C57BL/6 mice by regulating the level of 4-HNE following TPA treatment. We will further characterize the Psl1.2 locus to determine if any other genes mapping within the locus are modifiers of TPA skin tumor promotion susceptibility. In addition, we will identify and characterize genes mapping to Psl1.1 and Psl1.3 that are associated with responsiveness to TPA-induced skin tumor promotion in DBA/2 and C57BL/6 mice. The Specific Aims are: i) To further characterize the role of Gsta4 as a modifier of TPA skin tumor promotion susceptibility; ii) To characterize the mechanism for, and consequences of, strain-specific induction of Gsta4; and iii) To identify and characterize genes mapping within the Psl1.1 and Psl1.3 loci that modify the response to TPA skin tumor promotion. PUBLIC HEALTH RELEVANCE: The primary goal of the research proposed in this grant application is to identify and characterize genes that modify susceptibility to skin tumor promotion using the multi- stage skin carcinogenesis model in mice. Data generated from these studies will lead to identification of genes that regulate susceptibility to cancer in human populations and the development of novel cancer prevention strategies.
DESCRIPTION (provided by applicant): Breast cancer is one of the leading causes of premature death in women and development of chemoprevention and chemotherapeutic interventions wilt require understanding of the important steps that lead to tumor formation, growth and metastasis. Estrogen receptor (ER)-positive and -negative breast cancer cells have been extensively used as models for studying signaling pathways that are important for breast tumor growth. Both 17beta-estradiol (E2) and polypeptide growth factors (GFs) have been identified as important mitogens for breast cancer cell growth. We hypothesize that ERalpha-dependent activation of nongenomic phosphatidyl inositol 3-kinase (PI3-K) and mitogen-activated protein kinase (MAPK) pathways by E2 are important for breast cancer cell proliferation, and the proposed studies will investigate the mechanisms of non-genomic activation by E2. Aim 1 will focus on the role of ERalpha in activation of PI3-K and MAPK using ER-positive breast cancer cells (MCF-7 and ZR-75) and ER-negative (CHO and COS) cells as models. The domains of ERalpha required for activation of kinases and the importance of cell context will be determined in transient transfection studies. The mechanisms of non-genomic action of E2 and the role of specific kinases in mediating activation of PI3-K and MAPK will be investigated in Aim 2. The studies will also determine the biological significance of direct ERalpha interactions with Src-SH2, G proteins, the p85 regulatory subunit of PI3-K and the IGF-1 receptor. Aim 3 will focus on downstream mechanisms of ERalpha-dependent activation of c-fos by kinases. Dominant negative p85, MAPKK and Src expression plasmids transiently or stably transfected into breast cancer cells will be used to investigate the contribution of the MAPK and PI3-K pathways to the mitogenic activity of E2 in breast cancer cells (in vitro) and mammary tumors in athymic nude mice bearing breast cancer cell xenografts. The in vitro mechanistic studies will also be complemented by research on the contributions of kinase activation by E2 on cell proliferation and tumor growth and thereby define potential therapeutic targets for treating breast cancer.
Crisp Terms/Key Words: athymic mouse, biological signal transduction, cell growth regulation, estradiol, transfection, gene expression, protooncogene, growth factor, insulinlike growth factor, breast neoplasm, neoplastic cell, neoplastic growth, protein structure function, G protein, growth factor receptor, estrogen receptor, tissue /cell culture, enzyme activity, MCF7 cell, mitogen activated protein kinase, phosphatidylinositol 3 kinase, cell proliferation, fos protein, protein protein interaction
DESCRIPTION (provided by applicant): Toxins, pathogenic infection (viral and bacterial), and physical injury to the liver results in a loss of hepatic tissue, triggering a regenerative response to restore liver cell mass. Dysregulation in the repair process can lead to liver failure or liver cancer. The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor functionally identified with proliferative processes. The AhR ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) the prototype for a class of compounds responsible for a range of toxic or adaptive endpoints, inhibits liver regeneration following tissue injury thus implicating the AhR in liver repair. Our long-term goal is to understand mechanistically how the AhR contributes to liver homeostasis by regulating cell proliferation, and thereby identify the molecular basis for TCDD-induced disruption of normal biological processes. We hypothesize that the AhR plays an important role in liver homeostasis, in part by regulating progress through G1 phase of the cell cycle in proliferating hepatocytes. The goal of this proposal is to examine the functional relationship between AhR activity and TCDD responsiveness proteins known to regulate hepatocyte proliferation. Specifically, the planned studies will examine the molecular basis and relative contribution of cyclin-dependent kinase 2 (CDK2) inhibition and plasminogen activator inhibitor-1 (PAI-1) expression on liver regeneration. TCDD alters CDK2 activity and PAI-1 expression during liver regeneration, representing distinct intracellular and extracellular (autocrine or paracrine) mechanisms of action that conspire to inhibit the restorative response to injury. Preliminary results also demonstrate that AhR regulation of the PAI-1 gene involves a novel (non-XRE) DNA element that will be characterized in detail. The proposed studies are an extension of currently funded research looking at the AhR in regulating hepatic proliferation in cell culture models, and seeks to build on previous findings using a physiologically relevant (non-transformed) model of cell proliferation. Parallels between humans and mice in the regenerative response, instills confidence that findings generated during these studies will be directly applicable to the human condition.
Crisp Terms/Key Words: protein protein interaction, aromatic hydrocarbon receptor, retinoblastoma protein, gel mobility shift assay, toxin metabolism, environmental toxicology, receptor expression, receptor binding, DNA binding protein, protein structure function, protein sequence, toxicant interaction, dioxin, complementary DNA, liver regeneration, immunoprecipitation, western blotting, immunocytochemistry, molecular cloning, carbopolycyclic compound, polymerase chain reaction, cell growth regulation, laboratory rat, laboratory rabbit
DESCRIPTION (provided by applicant): Dioxin and related halogenated aromatic hydrocarbons are ubiquitous, persistent environmental contaminants causing adverse responses to human and wildlife. Most of the toxic responses induced by dioxin are mediated by the aryl hydrocarbon receptor (AhR). Therefore, central to our understanding of dioxin-induced toxicity is to elucidate the mechanism of the AhR-regulated gene expressions. In earlier studies, we have found a physical association and functional reciprocal repression between the AhR and NF-kB pathways (J. Biol. Chem. 274,510). Because NF-kB is a pleiotropic transcription factor involved in many physiological functions that are known to be adversely affected by dioxin, the AhR-mediated suppression of NF-kB offers a mechanism for some aspects of hitherto poorly understood dioxin-induced toxic responses, such as the immune suppression and abnormal skin proliferation. Reciprocally, suppression of AhR by NF-kB activation has also offered an underlying mechanism for the long-standing observation that inflammatory cytokines and lipopolysaccharide suppress AhR-regulated cytochrome P450 1A1/1A2 and decrease capacity of xenobiotic (including clinical drugs) metabolism (J. Biol. Chem. 276,39638). In recent studies, by using chromatin immunoprecipitation (CHIP) assay, we have obtained new results revealing that the AhR/NF-kB interaction converges at level of transcription involving (1) control of transcription elongation and (2) chromatin modifications. In AIM I of this proposal we will investigate a mechanism in which AhR/NF-kB interaction regulates cyp1a1 transcription elongation by directly interacting with p-TEFb (positive transcription elongation factor b), which plays a critical role in elongation control. In AIM II, we will investigate histone modifications (histone acetylation and methylation) in response to the diametrically opposing actions of AhR and NF-kB and to establish the residue-specific and combinatorial patterns of histone modifications (histone code) associated with "on and off" states of cyp1a1. We will also investigate a novel AhR interactive protein (identified by CytoTrap yeast two hybrid screening) SUV39H2 methyltransferase for its role in AhR-mediated gene silencing, which may be important for male imprinting. The proposed studies will help us gain mechanistic understandings of the functions of AhR and NF-kB in normal physiology as well as pathogenesis induced by dioxin and related compounds.
Crisp Terms/Key Words: protein protein interaction, yeast two hybrid system, aromatic hydrocarbon receptor, nuclear factor kappa beta, SDS polyacrylamide gel electrophoresis, methyltransferase, environmental toxicology, histone, lipopolysaccharide, phosphorylation, dioxin, immunoprecipitation, cytokine, gene induction /repression, transcription factor, gene expression, polymerase chain reaction, biological signal transduction
DESCRIPTION (provided by applicant): Polycyclic aromatic hydrocarbons (PAHs), which are present in tobacco smoke and in charcoal broiled meats, cause cancers in laboratory animals and are suspected human carcinogens. The central hypothesis of this application is that a cytochrome P4501A2 (CYP1A2)-dependent) metabolite of 3-methylcholanthrene (MC) plays an important role in the sustained induction of CYP1A1, a phenomenon that may have important implications for carcinogenesis. We propose the following specific aims. 1. To investigate the molecular mechanisms of sustained induction of CYP1A1 by MC, and test the hypothesis that a CYP1A2-dependent metabolite of MC contributes to the sustained CYP1 Al induction by MC. Wild-type (WT) and CYP1A2- knockout (KO) mice will be treated with MC (100 fmol/kg), once daily for 4 days, or a single dose of MC (100 fmol/kg) and at selected time points, levels of ligand-bound Ah receptor (AHR) in hepatic or pulmonary nuclei will be determined by electrophoretic mobility shift assay (EMSA)/Western blotting. 2.To test the hypothesis that CYP1A2-dependent metabolism of MC in hepa-1 cells or in mice leads to formation of sequence-specific DNA adduction, as determined by ligation-mediated polymerase chain reaction (LM-PCR), on the regulatory regions of the CYP1 Al promoter [e.g., AHR response elements (AhREs)], leading to a novel mechanism by which MC-DNA adducts will upregulate CYP1A1 gene. 3. Using a transgenic mouse line carrying the human CYP1A1 promoter, the hypothesis, that persistent induction of CYP1A1 expression by MC occurs in a tissue-specific manner, and is preceded by sustained activation of the CYP1A1 promoter, will be tested. 4. To test the hypothesis that CYP1A2-null mice will be more susceptible to MC-induced pulmonary carcinogenesis than similarly exposed WT mice. WT or KO mice will be exposed to MC, and CYP1A1 expression, DNA adducts, alteration in the expression of other cancer-related genes, as determined by cDNA microarray analyses, and tumor histology will be studied. The long-term goals are to: (i) define the molecular mechanisms of regulation of CYP1A1 gene expression by PAHs and (ii) develop rational strategies for the prevention/treatment of humans cancers caused by environmental chemicals.
DESCRIPTION (provided by applicant):
Chromosomal translocations resulting in inappropriate control of disease-related genes are important causative factors in environmentally induced disorders such as cancer. For example, translocations resulting in overexpression of the BCL-2 and c-MYC genes are the hallmarks of follicular B-cell lymphoma and Burkitt's lymphoma, respectively. DNA double-strand breaks (DSBs) are the first step in the process of chromosomal translocation. However, little is known about the mechanism(s) of the breakages on the translocated genes, why the DSBs tend to locate in certain genomic fragile site "hotspots", and the effects of environmental agents on the genomic instability at these susceptible hotspots. In this application, the intent is to use a comparative genetic approach to determine mechanisms of DNA structure-induced genomic instability. Interestingly, the most common breakpoints in such genes occur near regions that are capable of adopting non-B DNA structures. This group has discovered that H-DNA and Z-DNA-forming sequences near the translocation breakpoint hotspots in the human c-MYC gene induce DSBs, resulting in high levels of genetic instability in mammalian cells. Hence, the objectives of this application are to determine the mechanisms involved in genetic instabilities at breakpoint hotspots associated with disease, and further the development of novel approaches to reduce genetic instability caused by environmental DNA damaging agents. The immediate goals are to test the hypotheses that non-B-DNA structures found in the BCL-2 and c-MYC breakpoint regions are implicated in genetic instability across species, and that DNA damage induced by environmental factors at these hotspots enhance their susceptibility to genomic instability. The following are proposed: 1) to measure non-B DNA-induced genetic instability in different species. The naturally occurring H-DNA or Z-DNA-forming sequences from the human c-MYC and BCL-2 genes will be screened for their mutagenic potentials in a variety of species including yeast, mouse, and human. DNA structure-induced DSBs, illegitimate recombination, or point mutations in cells will be detected by facile blue/white screening; 2) to determine the susceptibility of non-B DNA-sequences to environmental carcinogen-induced DNA damage and mutagenesis. Environmental carcinogens such as irradiation are known to result in more non-B structure formation, and a reduced error-free repair of the damage. Thus, the amount of DNA damage induced and repaired in non-B sequences will be measured, and the level of genetic instability induced by environmental agents at these fragile site "hotspots" determined; and 3) to identify the genes/pathways that are involved in the genetic instability at non-B DNA sequences in the presence and absence of environmental carcinogens. Using a high-throughput screen gene products involved in DNA structure-induced genetic instability will be identified at genomic hotspots to begin to elucidate the pathways involved in genetic instability. Data obtained will give a better understanding of the mechanisms of genomic instability and the impact of environmental agents on these mechanisms. These discoveries should begin to unravel the pathogenesis of diseases that are caused by genomic instability, and ultimately to the development of new approaches for treatment and prevention.
DESCRIPTION (provided by applicant):
The primary goal of the research proposed in this application is to identify and characterize common genes, pathways, and networks that are involved in tumor promotion by diverse promoting stimuli. The carcinogenic process following exposure to chemical carcinogens occurs via a multi-step process involving initiation, promotion, and progression. Previous work from our laboratory using a genetic approach has provided evidence that there are common pathway(s) controlling susceptibility to tumor promotion by diverse tumor promoting stimuli including chemicals and wounding. Studies of the susceptibility to skin tumor promotion by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) have revealed that it is a multigenic trait, and we have mapped loci that modify TPA promotion susceptibility to several chromosomal regions. Using omics" approaches, we have recently identified genes that map to some of these regions that may play a role in this susceptibility (i.e., Gsta4 on chr 9, and S100A8 and S100A9 on chr 3). Additional preliminary data suggest that changes in expression of Gsta4, a gene mapping to one region, may be a common event in tumor promotion by several classes of compounds. These data suggest that an approach to uncover genetically conserved pathways could lead to discovery of common mechanisms underlying an important process (i.e., tumor promotion) in carcinogenesis. In this proposal, we will use a genetical genomics strategy to test the hypothesis that diverse classes of compounds promote the development of skin tumors through common pathways. Genes associated with these pathways will be characterized to confirm their role in the mechanism of skin tumor promotion. The specific aims of this proposal are 1) determine the strain distribution patterns (SDP) among BxD recombinant inbred (Rl) strains for susceptibility to TPA, okadaic acid (OA), and chrysarobin (Chry) skin tumor promotion, 2) examine global mRNA and protein expression profiles in epidermis of BxD Rl strains to identify common gene pathways and networks associated with tumor promotion, and 3) examine the role of Gsta4 and other candidate genes/pathways in the process of tumor promotion. The research proposed in this application will make it possible to identify new targets for cancer prevention and will likely make it possible to identify individuals with a high risk for tumor development.
DESCRIPTION (provided by applicant): AP-endonuclease (APE) plays a central role in repair of most genomic damage induced endogenously and by environmental agents that induce oxidative stress. The mammalian APE, (APE1/Ref-1) additionally functions in transcriptional regulation: both as a reductive activator of transcription factors involving Cys 65 (or possibly another Cys residue) and directly, as a trans-acting co-factor (in acetylated form) in repressing negative Ca2+ response element (nCaRE)-dependent parathyroid, renin, and possibly other genes. Acetylated APE1 also binds to shear stress response element (SSRE), present in PDGF, eNOS and other shear-activated genes in endothelial cells. APE1 does not directly bind to a cis element, but to other proteins present in trans-acting complexes. Furthermore, APE1's stable binding to the Y-box specific trans-acting factor YB-1 suggests its role in regulation of even other genes including p53. We have generated conditional mouse embryo fibroblasts (MEF) lacking endogenous APE1 alleles but expressing human APE1 transgene; deletion of the transgene induces apoptosis of MEF, which showed that APE1 is essential for somatic cells as well as for the mouse embryo. Prevention of apoptosis by providing exogenous APE1, mutated to inactivate its repair function or its acetylation sites indicates essential roles of both functions. Regulatory activity of APE1 for cell survival is also suggested from its frequent overexpression in cancer cells. Furthermore, synergy between APE1 and p53 in spontaneous tumor induction in mice implicates APE1 in cancer prevention. In spite of extensive documentation of APE1's diverse regulatory functions, the requirement of its specific sequence motifs, domains and active sites for regulatory functions in various systems has not been clearly defined. Our central hypothesis is that APE1, in addition to carrying out essential repair of endogenous genome damage, regulates as a co-activator or co-repressor of genes which are essential for survival or for maintaining homeostasis after exposure to stress. To test this hypothesis and to elucidate APE1's structure- function requirements for the regulatory activities we will pursue three aims: (i) to define the requirements of APE1's specific residues and motifs for preventing apoptosis in MEF mutants by complementation; (2) to characterize APE1's interaction with other trans-acting factors present in nCaRE-, SSRE-, and Y-bound-box complexes chosen as prototype systems; and (3) to confirm the regulatory functions of APE1 for a few apoptosis-linked genes identified by preliminary gene chip screening. The results obtained from these studies will provide a comprehensive picture of the molecular bases for APE1's diverse regulatory activities. Furthermore, our studies should help identify APE1-regulated key signaling pathways in tumor induction and promotion which could be potential targets for cancer therapy.
DESCRIPTION (provided by applicant): Prokaryotes and eukaryotes are endowed with multiple specialized DNA polymerases that support DNA synthesis across sites of DNA damage that arrest normal high-fidelity replication. This process is called translesion DNA synthesis (TLS). This function is fundamental to the survival of cells that have suffered arrested DNA replication due to the presence of unrepaired DNA damage, and to the genesis of mutations in living cells. These polymerases may also play a role in somatic hypermutation (SH) in the immune system by filling in gaps in DNA in an error-prone manner, increasing the diversity of antibodies. Understanding the molecular mechanism of TLS is therefore central to understanding all human diseases associated with mutagenesis, especially cancer, and to understanding SH in the immune system. We have identified a polymerase, POLK, and created a mouse model for this damage inducible polymerase. The progeny of Polk-/-mice manifest a mutator phenotype and cells from these mice are sensitive to killing by UV radiation and to the polycyclic aromatic mutagen and carcinogen benzo[a]pyrene. Specific aim 1 of this renewal proposal is to characterize the molecular basis of the mutator phenotype identified in progeny of Polk-/-mice by using mutation detection systems to clarify the in vivo functions of POLK. These systems will identify changes found in the DNA of mice lacking this enzyme. Aim 2 is to identify the functions of the multiple POLK isoforms present in mouse testis, their biochemical interactions with other proteins and DNA as well as their ability to add nucleotide opposite DNA lesions. We have also identified proteins that interact with POLK and are pursuing how these interactions affect damage recognition and repair. Aim 3 is to elucidate the molecular mechanism(s) of DNA polymerase recruitment during TLS through the detailed characterization of REV1 protein in mammalian cells and in the yeast S. cerevisiae, a genetically tractable model eukaryote. The results of these studies will clarify the in vivo functions of POLK and lead to further understanding of how POLK and it's protein isoforms interact with REV1L during polymerase switching will help define how this aspect of genome maintenance works to prevent mutations that lead to cancer and other diseases.
Crisp Terms/Key Words: protein protein interaction, neoplastic process, protein isoform, enzyme activity, cell line, phenotype, DNA damage, DNA directed DNA polymerase, DNA repair, DNA replication, polymerization, biological model, immunogenetics, gene mutation, mutagen, Saccharomyces cerevisiae, enzyme induction /repression, pathologic process, molecular dynamics, genetically modified animal, laboratory mouse
DESCRIPTION (provided by applicant):
Nuclear hormone receptor activity is regulated by accessory proteins such as histone acetyltransferases and arginine methyltransferases, that function as coactivators for these receptors and such are involved in hormone responsive tumor progression. This activity is facilitated in part by the ability of the protein arginine methyltransferases (PRMTs), PRMT1 and CARM1, to methylate histones in vivo and these coactivators are potential targets for drug development. Xenoestrogens are compounds of plant or synthetic origin that often share structural similarity with the principal mammalian estrogen, 17beta-estradiol. Recently, a high throughput screen for small-molecule inhibitors of the PRMT sub-class of coactivators was performed. The majority of these inhibitors were polyphenols, and one in particular (AMI-18) shared additional features with a group of xenoestrogens. Thus a panel of xenoestrogens was tested and a large number of them found to inhibit PRMT activity in vitro. In this study, the intent is to evaluate the ability of xenoestrogens and SERMs to regulate methyltransferase and acetyltransferase activities of recombinant proteins and in cells. Techniques to be used will include in vitro methylation assays, transcriptional reporter assays, chromatin precipitation (ChIP) experiments and RT-PCR. In this exploratory/developmental research proposal the novel idea that some xenoestrogens posses the ability to both bind the estrogen receptor (ER) (thus perturbing transcription) and inhibit co-activator activity (thereby suppressing transcription) will be investigated. This raises the possibility that combination treatment with the best antagonists the best co-activator inhibitors may be of therapeutic value particularly for hormone-dependent tumors, like breast and prostate cancers. The specific aims are to characterize: 1) Biochemically xenoestrogens as co-activator inhibitors; and 2) In vitro xenoestrogens as co-activator inhibitors.
DESCRIPTION (provided by applicant): Rev1 is unique among DNA polymerases in that the protein itself rather than the DNA template determines the specificity for both the templating and the incoming nucleotide. The Rev1 crystal structure that we have solved suggests an elegant mechanism by which this polymerase could promote proficient and error-free replication through a large variety of N2-adducted guanines that result from endogenous oxidative damage and from exposure to a number of widespread DNA damaging chemical and environmental carcinogens such as butadiene epoxides and anti-benzo[a]pyrene diol epoxides. Such a role for yeast Rev1 will be examined using a combined biochemical, genetic, and structural approach. In Aim 1, key amino acid residues involved in the pairing with the incoming dCTP and in the eviction and stabilization of templating G will be mutated and their effects on nucleotide incorporation specificity and catalytic efficiency determined. In Aim 2, the conformational changes that occur in Rev1 upon DNA binding and upon dNTP binding will be analyzed through crystal structures of the Rev1 apoenzyme and Rev1.DNA binary complex and their comparison to the structure of Rev1.DNA.dCTP ternary complex. In Aim 3, biochemical studies will be undertaken to test the hypothesis that a major role of the Rev1 DNA synthetic activity is to promote efficient and error-free replication through various N2adducts of guanine that sterically impinge upon the minor groove, and which result from cellular oxidative damage or from exposure to DNA damaging environmental carcinogens. Also as part of this aim, we will examine the means by which complex formation between Rev1 and the extender polymerase coordinates the nucleotide insertion and the subsequent extension steps in the bypass of these adducts. As a complement to these biochemical studies, in Aim 4, crystal structures of Rev1 with DNAs containing a variety of N2 guanine adducts will be determined, as well as the structure of Rev1 with an abasic lesion. In Aim 5, genetic studies will be done to establish the requirement of the Rev1 DNA synthetic activity in promoting error-free replication through the various N2-adducts of guanine in yeast cells. Rev1 as well as the other DNA repair proteins are highly conserved between yeast and humans. The proficient and accurate ability of Rev1 for promoting replication through the large variety of DNA adducts that form at the N2 of guanine will have a major impact on genome stability by keeping the rate of mutations low, reducing thereby the incidence of carcinogenesis in humans. The results of this study are highly relevant for cancer biology and etiology, as error-free replication through DNA lesions provides for an important means of cancer prevention. PUBLIC HEALTH RELEVANCE: DNA lesions generated from cellular oxidative damage and from exposure to environmental pollutants affect the stability and integrity of genomic DNA. Error-free replication through such lesions reduces their adverse impact by keeping the rate of mutations low and by reducing the incidence of cancer formation. The proposed studies will examine the role of Rev1 DNA polymerase in promoting error-free replication through DNA lesions.
DESCRIPTION (provided by applicant): Translesion synthesis (TLS) DNA polymerases (pols) promote replication through DNA lesions. Humans possess four TLS pols that belong to the Y-family, Pols eta, iota, kappa, and Rev1, and another Pol, Pol zeta, that belongs to the B-family. The overall objectives of this proposal are to elucidate the biological roles of these pols in TLS in human cells, to determine whether they function in an error-free or mutagenic manner, and to identify the means by which a TLS Pol gains access to PCNA and thereby to the replication fork stalled at a DNA lesion site. These and related questions will be studied in the following Specific Aims. In Aim 1, the roles of TLS Pols in promoting replication through a diverse set of DNA lesions in human cells will be analyzed using an SV-40 based plasmid system that we have constructed. DNA lesions to be studied include UV photoproducts, and DNA adducts such as 1,N6-ethenodeoxyadenosine, 1,N2-propano-2'-deoxyguanosine, and (+) trans-dG-N2- benzo[a]pyrene diol epoxide, which result from cellular oxidative damage and from exposure to DNA damaging environmental carcinogens. In Aim 2, studies will be done to test the hypothesis that in human cells, Rev1 functions as a structural element for Pols eta, iota, and kappa. In Aim 3, the roles of TLS Pols in promoting replication through UV lesions and whether they do so in an error-free or mutagenic manner will be studied in the chromosomal cII transgene carried in a mouse embryonic fibroblast cell line. The contributions that cyclobutane pyrimidine dimers vs. (6-4) photoproducts make to UV mutations resulting from the action of different TLS Pols will be analyzed. In Aim 4, the model that, in addition to their binding to PCNA via their PIP domain, TLS Pols bind the ubiquitin moiety on PCNA via their ubiquitin binding domain, will be tested by genetic and biochemical studies of mutations in these domains of Pol eta. In Aim 5, biochemical studies will be undertaken to test the hypothesis that upon stalling at a DNA lesion site, the binding of the replicative pol to PCNA is attenuated as a result of PCNA ubiquitination, and that, in turn, promotes the access of the TLS pol to the primer-template junction via its binding to ubiquitinated PCNA (Ub-PCNA). Specifically, these studies will examine how Ub-PCNA promotes synthesis by Pol eta through a cis-syn TT dimer when a processively moving Pol delta has become stalled at the lesion site. By helping ensure the continuity of the replication fork, TLS Pols play an important role in the maintenance of genomic integrity. Furthermore, their proficient abilities for promoting error-free replication through a large variety of DNA adducts that result from cellular oxidative reactions and from exposure to chemical and environmental carcinogens will have a major impact on genome stability by keeping the rate of mutations low, reducing thereby the incidence of carcinogenesis in humans. In fact, the inactivation of Pol eta in humans results in highly elevated levels of skin cancers. The proposed studies are highly relevant for cancer etiology as the results will reveal how human cells minimize the mutagenic and carcinogenic consequences of DNA lesions. PUBLIC HEALTH RELEVANCE: DNA lesions are generated in human cells from cellular oxidative reactions and from exposure to a variety of environmental pollutants and carcinogens. By promoting error-free replication through such DNA lesions, translesion synthesis DNA polymerases would play an important role in maintaining genome stability by keeping the rate of mutations and hence the incidence of cancers low. The proposed studies will elucidate the roles of human DNA polymerases in promoting replication through a variety of DNA lesions and they will examine the mechanisms of this process.
DESCRIPTION (provided by applicant):
The objectives of the proposed study are to: 1) estimate the geographic
distribution of environmental lead in El Paso and Juarez; 2) estimate the
prevalence of elevated blood lead levels in the two cities; 3) differentiate
chronic and acute lead exposures in children with elevated lead levels;
4) fractionate lead by etiological source in children; and 5) involve end-
users to evaluate a scientifically based, culturally relevant
prevention/intervention strategy. The study seeks to improve the capacity of
the El Paso, Texas - Juarez, Mexico binational community to participate in
research to identify the etiology of lead exposure among low-income Hispanic
children. Project activities will increase the community's capacity to
deliver environmental health intervention, prevention, and educational
services in a binational setting through collaborative partnerships between
academic institutions, community organizations, and stakeholders. Based on
original research, the study joins environmental health scientists and
community organization members in an interdisciplinary investigative team.
This project will evaluate lead exposure and its adverse effect on the health
of low-income Hispanic children on the U.S.-Mexico border. Environmental lead
distribution will be estimated using data from indoor and outdoor sites. Nine
hundred and thirteen blocks will be tested for outdoor lead levels in each
city. In each block 40 random samples of soil will be analyzed using a
portable X-ray fluorescence (XRF) lead analysis unit in public areas along
streets, from playgrounds, schools, hospitals, etc. Indoor lead levels in
water, soil, dust, and air will be tested in 4 households in 10 randomly
selected blocks. An exposure assessment survey will be administered. High-
household's risk will be defined as those exceeding EPA maximum household
contaminant levels. Data will be statistically analyzed to produce binational
maps of environmental lead distribution.
Two hundred households will be randomly selected from high-risk blocks in each
city (a total 400 households). One hundred and ninety-two children, ages 6
months to 11 years, will be recruited in each city. Blood lead levels will be
analyzed using graphite furnace atomic absorption spectroscopy. Children with
elevated lead levels (>10 ug/dL) will be tested to determine whether the
exposure is chronic using K X-ray fluorescence spectroscopy of the tibia. The
etiological source of lead in the blood will also be evaluated by inductively
coupled plasma - mass spectroscopy (ICP-MS). Regression analysis will be
conducted to model the relationships between blood and bone lead levels,
environmental lead, and risk of exposure. Four community-based organizations
(CBOs) will participate in the research process. A CBO liaison will build and
coordinate a community-based partnership for education and outreach and
overall capacity building.
DESCRIPTION (provided by applicant):
Many of the most challenging contaminated sediment sites awaiting effective remediation exhibit separate phase contamination (nonaqueous phase liquids or NAPLs), or mobile aromatic or chlorinated solvents. Conventional sediment remedial approaches, including dredging and capping, are not designed nor effective for the management of fluid phase contaminants. The use of a low permeability cap provides opportunities to control contaminant migration and simultaneously introduce treatment in the underlying sediment without loss of reagent or contaminant. Further, by maintaining hydraulic control, the contaminants or the treated residual can be funneled to a relatively small area, or "gate", for further treatment of any residual contamination. The proposed research is designed to develop this novel remedial approach that is an adaptation of proven funnel and gate technologies for groundwater control. Specific treatment technologies to be evaluated and developed include the use of organoclays for the sorption and stabilization of NAPLs, zero-valent iron for the reductive dechlorination of chlorinated solvents and enhanced biological treatment for the control of both aromatic and chlorinated solvents. The project will test the broad hypotheses that A funneling cap can effectively contain contaminants as well as introduced reagents beneath the cap and effectively channel interstitial fluids (water or NAPL) to a collection or treatment gate Slurries of treatment media, specifically organo-modified clays for NAPL stabilization, zero valent iron for encouragement of reductive dechlorination, or reagents encouraging biological degradation, can be introduced beneath a low permeability cap and stabilize or encourage reductive treatment of non aqueous and aqueous contaminants Treatment gates composed of reactive materials or encouraging biological degradation, either alone or in combination, can effectively manage residual contamination from the in-situ treatment zone The primary goal of the proposed research is control of exposure and risk to human health and the environment at the sediment-water interface. The resulting reduction in exposure and risk to benthic and aquatic organisms will eliminate human food chain exposure to contaminants in sediments.
DESCRIPTION (provided by applicant): The primary objective of the Center for Research on Environmental Disease (CRED) is to study the mechanisms by which environmental factors may cause or influence human disease and to develop methods for early detection, prevention, and control of environmentally related diseases. The theme for the CRED is to define molecular pathways that lead from environmental exposures to adverse outcomes and to understand the genetic basis for variability in these responses. In the next funding period, CRED members will continue to focus a major research effort on developing and using animal models of human environmental diseases, especially cancer. In addition, the Center will expand research efforts in the area of disease prevention by focusing a greater effort on energy balance and its influence on environmental disease risk. Finally, these research efforts will again be complemented and aided by studies of genetic risk factors in diverse human populations. These areas of focus are selected because they represent major research themes that cut across more than one Research Core. Because the problems of environmental health are complex, requiring an understanding of the sources of chemical and physical stresses in the environment, their modes of transport and transformation, the routes of human exposure, the mechanisms through which the agents exert their effects, and the possible ways their actions may be influenced by modifiers or co-factors, including genetic background, the successful study of these complex problems requires interdisciplinary approaches. To tackle this vast array of problems, the CRED brings together, in an integrated effort, a multidisciplinary group of established scientists with an extremely broad range of expertise. In addition, the CRED will continue to provide these investigators access to sophisticated biochemical, molecular, and analytical techniques to enhance research efforts that revolve around the Center theme. This Center consists of five Research Cores: i) Mechanisms of Toxicity and Cell Death; ii) Cellular Responses to DNA Damage; iii) Molecular Genetics and Environmental Carcinogenesis; iv) Molecular Epidemiology and Ecogenetics; and vi) Targets and Mechanisms of Disease Prevention. The research efforts of these Research Cores will be enhanced by access to the following six Facility Cores: i) Molecular Biology; ii) Transgenic Animals; iii) Histology and Tissue Processing; iv) Cell and Tissue Analysis; v) Analytical Instrumentation; and vi) Biostatistics and Informatics. In addition, an Administrative Core formalizes collaborative interactions and provides for enrichment activities as well as a Pilot Project Program. Finally, a Community Outreach and Education Program also establishes a mechanism to disseminate important research findings of the Center to the general community, as well as provides community education programs with an emphasis on issues related to environmental health sciences.
DESCRIPTION (provided by applicant)
This application requests continued support for the NIEHS Center at the University of Texas Medical Branch. The mission of this Center is to integrate, coordinate and foster interactions and collaborations among a group of established investigators pursuing research pertinent to the effects of environmental factors on human health. The proximity to sources of many significant environmental problems, such as ozone pollution, emissions of fine particulates, hazardous chemical releases, hazardous waste sites, and pediatric lead exposure, makes UTMB a compelling site for a multidisciplinary environmental health sciences center. During the nine years of its existence, this Center has emerged as a national leader in elucidating cellular response mechanisms to environmental challenge and in working with the community to enhance awareness of environmental health issues and elaborate prevention and intervention strategies. The overarching theme of the UTMB NIEHS Center is the role of oxidative stress in mediating the health effects of exposure to environmental factors. Center investigators are aided by four service cores (Molecular Genetics, Biomolecular Resource Facility, Cell Biology, and Synthetic Organic Chemistry), which provide advanced technologies, unique reagents, and specialized expertise, as well as cost-effective and efficient access to more routine services or research materials. Scientific findings from the Center are communicated to the public through a vibrant COEP with advice from a Community Outreach Board.
The Center for Environmental and Rural Health (CERH) at Texas A&M University was created to support state-of-the-art research in the environmental health sciences and to translate this knowledge into concepts and principles that can be easily adopted by rural communities in the State of Texas and beyond. The CERH provides administrative infrastructure and technical support to foster multidisciplinary research into basic mechanisms of environmental disease, from the gene to the organismal level, from molecules to social factors. The research activities of Center investigators have been clustered into four major areas: Biostatistics and Community Health, Chemical Biology, Nutrition, and Reproductive and Developmental Biology. Each of these Research Cores consists of senior and junior investigators representing multiple departments and programs throughout the University. The funded research of these investigators is enhanced by six Facility Cores that provide resources to assist with experiments involving the use of Analytical, Biostatistics and Computational, Genomics and Bioinformatics, Imaging, Protein Chemistry, and Transgenic technologies. The Pilot Project Program supports up to 5 exploratory research projects per year related to the research focus of the Center. A Community Outreach and Educational Program provides a mechanism for dissemination of important research findings of CERH investigators to rural communities, especially in the Lower Rio Grande Valley.
Crisp Terms/Key Words: biomedical facility, environmental health, rural area
DESCRIPTION (provide by applicant)
The proposed SBRP at Texas A&M University will utilize an integrated research model to investigate the potential adverse ecological and human health effects of Superfund chemicals and mixtures to improve the risk assessment paradigm. The major objective of the Program is to conduct fundamental research to reduce the uncertainty associated with risk assessment of complex mixtures, and to improve scientific and public confidence in the overall risk assessment process. The applicants hypothesize that adverse environmental and human health effects resulting from exposure to Superfund-related chemical mixtures are multi-factorial and associated with interactions of individual chemicals and their disposition, as well as exposure of target biological receptors and their genetic sensitivities. The proposed studies include three mechanism-based research projects focused on Superfund toxicants that act through endocrine disruption, genotoxicity and cellular injury. Project 4 will investigate genetic susceptibility and predisposition to chemical-induced birth defects. The development of novel methods for remediation of contaminated media will be carried out in Project 5. Human and ecosystem exposure and risk assessment studies will be investigated in Project 6. Project 7 will investigate gene-environment interactions affecting the risk of birth defects. Research in the seven individual projects will be supported by the Image Analysis, Analytical, Field Studies, Administrative, and Training Cores. The Program also includes a Research Translation Core that will expand ongoing collaborations with Regional Site Managers at the United States Environmental Protection Agency (USEPA) to facilitate incorporation of biological test protocols into risk assessment at Superfund sites. An Outreach Core will communicate research results to community groups and students.
DESCRIPTION (provided by applicant): Congenital defects remain the greatest contributor to infant mortality; yet, the causes for the majority of these defects are either unknown or poorly understood. Amine- and amide-containing (nitrosatable) drugs and other compounds react with nitrite in the stomach to form N-nitroso compounds. In animal models, N-nitroso compounds have been found to induce a variety of congenital malformations. The few epidemiologic studies conducted in the past have focused on the separate effects of nitrates, nitrites, and nitrosatable drugs on risk of congenital malformations without consideration of their interaction in the endogenous formation of N- nitroso compounds. This case-control study will examine the separate and joint effects of prenatal exposures to nitrates, nitrites, and nitrosatable drugs on risk of selected congenital malformations (neural tube defects, limb malformations, oral clefts, and heart defects). Cases and controls will be obtained from the National Birth Defects Prevention Study (NBDPS), a multi-center study that covers populations in 10 different states. Subjects' usual intake of dietary nitrates, nitrites, and nitrosamines will be calculated from the NBDPS food frequency questionnaire. From the NBDPS interview, medications reported taken one month preconception and during the first trimester will be classified as to their likelihood of nitrosatability based on the literature and chemical structure. The periconceptional addresses of Iowa and Texas participants will be linked to community water systems and pertinent water nitrate sampling results. For Texas participants on private wells, we will model and predict nitrate levels with a multi-dimensional flow and transport model. We will analyze the separate and joint effects of nitrosatable precursors on risk of the selected malformations. We will also examine the effects of vitamins C and E (inhibitors of nitrosation) on the relations between nitrate/nitrite intake and nitrosatable drugs and risk of selected congenital malformations. Use of over-the- counter medications is fairly common during pregnancy according to a recently published study. Several over-the-counter preparations contain nitrosatable compounds as active ingredients. The proposed study will help us understand whether pregnant women who take these types of drugs and also consume greater amounts of nitrates and nitrites are at increased risk of having offspring with birth defects. The study is also designed to examine whether higher intakes of vitamin C or E decrease these potential risks.
DESCRIPTION (provided by applicant): Endocrine-disrupting chemicals such as environmental estrogens contaminate our surroundings and impair the reproductive health of animals, and probably humans. These compounds may act as inappropriate estrogens, and/or interfere with the actions of endogenous estrogens, but their mechanisms of action at low, environmentally relevant concentrations are largely unknown. We have recently shown that signal cascades leading to induced functions initiated by estradiol (E2) at the plasma membrane are also potently initiated by nonphysiological estrogens (xenoestrogens). In cells that express a membrane form of the estrogen receptor-a (mERa), each xenoestrogen elicited unique signaling patterns (temporal, dose-response) via activation of extracellular-regulated kinases (ERKs) and/or calcium elevation. We will now address (1) the structural requirements for activating mERa to generate signals and their linked functions, by comparing the effects of alkylphenol xenoestrogens having varying carbon- chain lengths and structures, and prominent physiological estrogens (E2, estriol, and estrone); (2) active alkylphenols' ability of to act in combination with physiological estrogens via additive, synergistic, and antagonistic mechanisms; and (3) the G protein coupling of these responses. G proteins likely lie upstream of the signaling responses shown by our previous work and others'. We will now seek direct evidence for G protein subtype interactions with mERa via co-immunoprecipitation, use of specific inhibitors, and dominant-negative G protein subtype and decoy interaction peptide approaches. Changes in G protein coupling in response to both physiological estrogens and alkylphenol xenoestrogens will be examined. Our long-term objective is to use our established model system to answer a variety of detailed mechanistic questions about how specific structural features of different physiological estrogens and xenoestrogen subclasses affect actions through the nongenomic pathway and the mERa, and thereby disrupt endocrine processes. Health relevance: Knowing how environmental estrogens disrupt normal signaling and reproductive functions will enable design of new prevention and treatment strategies to deal with their toxicity. Demonstrating their low-dose effects will also guide re-evaluation of Federal regulations setting legal contamination limits for environmental estrogens. The extent and mechanisms by which environmental estrogens contribute to diseases of estrogen overexposure (eg. breast and pituitary cancers, infertility) must be understood so that exposures can be limited to safe levels.
Crisp Terms/Key Words: alkyl group, biochemistry, biological signal transduction, calcium flux, estradiol, estrogen, membrane activity, phenol, G protein, estrogen receptor, mitogen activated protein kinase, endocrine disrupting compound
DESCRIPTION (provided by applicant): Endocrine disruptive (ED) agents in ground corncob bedding and foods block male and female sexual behavior and cyclicity in the rat and stimulate breast and prostate cancer cell proliferation. These agents separate into two components (Peak I-CM and Peak II-CM) during HPLC. The mitogenic agents in Peak I-CM (THF-diols) were identified as an isomeric mixture of 9, (12)-oxy-10, 13-dihydroxystearic acid and 10, (13)-oxy-9, 12-dihydroxystearic acids which block male and female sexual behavior and cyclicity. The mitogenic agents in Peak II-CM (LTX-diols) were identified as an isomeric mixture of leukotoxin-diol (LTX-diol) and isoleukotoxin-diol (iLTX-diol) that will be assessed as EDs in the proposed studies. The objectives of the proposed research are to identify the most biologically active synthetic THF-diol and LTX-diol isomers, define the nature of their interaction in modulating endocrine function and breast cancer cell proliferation and define specific biochemical sites regulated by these compounds in these systems. The most active THF-diol and LTX-diol isomer will be isolated and identified separately (Specific Aim 1) and whether they act additively or synergistically to stimulate breast cancer cell proliferation (cell cycle transition and apoptosis) and modify male and female sexual behavior and cyclicity will be determined (Specific Aim 2). The concentrations of THF-diol and/or LTX-diol isomers in tissues and blood from rats given "ED" doses of the compounds will be quantified by GC/MS and whether LTX-diols are precursors to THF-diols in rats will be studied (Specific Aim 3). The abilities of THF-diol and LTX-diol isomers to promote dimethylbenz(a)anthracene (DMBA)-induced mammary tumors in rats (Specific Aim 4), and to stimulate the proliferation (cell cycle transition and apoptosis) of estrogen receptor (ER) positive (MCF-7) or ER-negative (MDA-MB-231 cells) breast cancer cells in vitro or in vivo (in nude mice) by modulating phospholipase A2 (PoA), cyclooxygenase (COX), lipoxygenase (LOX) and aromatase will be evaluated (Specific Aim 5). Leukotoxins are known to affect NO release and it is possible that LTX-diol and THFdiol isomers inhibit female sexual behavior (Lordosis) by disrupting nitric oxide (NO) dependent pathways controlling LHRH release. This will be evaluated (Specific Aim 6). If THF-diols and LTX-diols impact endocrine and cell regulatory pathways by controlling LHRH release and lipogenic products that control cell proliferation, they likely have a major impact on behavioral and reproductive response profiles and cancer growth rates in experimental animals. Over the last decade, linoleic acid has replaced stearic acid in our diet. As metabolites of linoleic acid, increased intake of THF-diols and LTX-diols may influence human health and development. The proposed studies represent a critical step in defining target pathways for ultimately determining the mechanism of action of these compounds.
Crisp Terms/Key Words: dimethylbenzanthracene, terminal nick end labeling, cell proliferation, nitric oxide, cytotoxicity, male, female, estrogen receptor, sex behavior, phospholipase A2, lipoxygenase, prostaglandin endoperoxide synthase, aromatase, carcinogen, breast neoplasm, chemical related neoplasm /cancer, intermolecular interaction, sex cycle, endocrine gland /system, pharmacokinetics, flow cytometry, mitogen, cell cycle, laboratory rat, athymic mouse, diol
DESCRIPTION (provided by applicant): Long term goals are to determine the mechanisms of nonclassical steroid actions mediated by a novel family of putative membrane progesterone (P4) receptors (mPRs) we recently discovered in human cells and their functional significance in health and disease. Nonclassical P4 signaling pathways mediated via mPRa and mPRb in human myocytes will be investigated. Preliminary studies suggest these mPRs are upregulated in human myocytes during labor and activate multiple inhibitory G-proteins and second messenger pathways, resulting in down-regulation of nuclear progesterone receptor (nPR) transcriptional activity as well other conditions favoring myometrial contraction at term. Therefore, the hypothesis that P4 acts via the novel putative mPRa and mPR(3 receptors in human myocytes to activate multiple inhibitory G-protein/second messenger pathways and to modulate nPR transcriptional activity will be tested. Aims are to: (1) Determine steroid binding characteristics and localization of human wild type and recombinant mPRa and mPRb. Binding of progestins to cell membranes from human myocytes in the presence or absence of siRNA for the mPRs, and to mammalian cells transfected with human mPRs will be examined; (2) Investigate coupling of the mPRs to G-proteins and their associated second messenger pathways. Coupling of G-proteins, their identities and identities of second messengers activated will be investigated in these mPR cell expression models and in myometrial biopsy tissues (3) Investigate functional domains of hmPRa for steroid binding and G-protein coupling. Receptor domains of mPRa required for ligand binding and G-protein coupling will be investigated by developing pharmacophore and receptor models, followed by site-directed mutagenesis and functional analyses. (4) Explore modulation of nPR transcriptional activity and co-activator functions via mPR-dependent pathways. Cross-talk will be investigated using several reporter assays and nPR phosphorylation and co-activator function by immunological methods. Preterm birth is a major medical problem, occurring with 12% of births, but the mechanism of a functional progestin withdrawal in humans resulting in the onset of labor is unclear, The present study will characterize previously unrecognized multiple signaling cascades initiated by progesterone activation of mPRs that are likely involved in functional progesterone withdrawal at term, shifting the balance from a quiescent state to a contractile one. Although many of the specific experiments were difficult to follow, it appears that the overall organization attacks the issue on a broad front, such that a lot of information about the receptors will be forthcoming. It could be argued that this is just the type of characterization that has been done with many receptors. However, it is important to establish these points for the mPR. In addition, the studies should provide indications of important regulatory events that might occur in human cells.
DESCRIPTION (provided by applicant): Our long-term objective is to elucidate the mechanisms of the splenic toxicity of aromatic amines, and to develop preventive and therapeutic strategies. Using aniline as a prototypic compound, studies in our previous funding period demonstrated that aniline exposure in rats leads to oxidative stress in the spleen, evident from increases in reactive iron, lipid peroxidation, protein oxidation, DMA oxidation, lipid-derived aldehyde-protein adducts and nitrotyrosine. These changes are accompanied by activation of the transcription factors NF-kappaB and AP-1, and up-regulation of fibrogenic and inflammatory cytokines. Morphologically, spleen showed vascular congestion, increased red pulp cellularity due to increased sinusoidal cells and fibroblasts, capsular thickening and fibrosis. Having established the important role of oxidative stress in the splenic toxicity of aniline, we will now direct our studies toward the aniline-induced oxidative stress pathways that lead to splenic fibrosis and tumorigenesis. We hypothesize that aniline-induced oxidative stress due to over-production of oxidants (ROS and RNS) activates redox-sensitive transcription factors (NF-kappaB and AP-1) leading to transcription of genes involved in fibrosis and cell growth regulatory pathways in the spleen. Furthermore, oxidative damage to DNA leads to gene mutations. These events, individually or in concert, lead to fibrosis and/or tumorigenesis in the spleen. This hypothesis will be tested by pursuing three specific aims: Aim 1 will elucidate the regulation of transcription factors NF-kappaB and AP-1, signaling mechanisms and related gene expression in the splenic toxicity of aniline. Aim 2 will examine the role of oxidative DNA damage and repair, malondialdehyde-DNA adducts, DNA methylation, and mutations in the tumor suppressor gene p53 in aniline-induced splenic toxicity. Aim 3 will investigate the contribution of reactive nitrogen species to splenic damage by characterizing iNOS regulation and the formation of nitrated proteins, and by examining the role of NO/ONOO in the regulation of NF-kappaB activation, cytokine expression and DNA damage. These studies will elucidate the mechanisms of aniline-induced splenic toxicity, and will be important in devising strategies to prevent toxicity, and in risk assessment of aniline and other structurally-related aromatic amines.
DESCRIPTION (provided by applicant): Environmental chemicals are implicated as causative agents of autoimmune diseases (ADs). Our long-term goal is to elucidate the role of lipid peroxidation-derived aldehydes (LPDAs) and reactive nitrogen species (RNS) in the development of ADs induced and/or exacerbated by chemical exposure. Increased production of reactive oxygen and nitrogen species (RONS) has been implicated in the pathogenesis of ADs, and a large number of chemicals are known to produce RONS. Correspondingly, increased lipid peroxidation and protein nitration are also reported in systemic ADs. We hypothesize that increased production of LPDAs and RNS causes structural alterations to endogenous macromolecules, including proteins, through covalent binding and/or oxidation, resulting in the formation of neoantigens. Following antigen processing, these neoantigens can elicit autoimmune responses by stimulating T and B lymphocytes, eventually leading to ADs such as systemic lupus erythematosus (SLE). This hypothesis will be tested through four specific aims to: 1) Delineate the link between oxidative stress and autoimmunity. This will be achieved by exposing autoimmune-prone (MRL+/+) and -resistant (B6C3F1) mice to trichloroethene (TCE, an environmental chemical known to cause lipid peroxidation/RNS). Formation of LPDA-protein adducts (in liver, kidney and spleen) and their corresponding antibodies will be correlated with autoimmune response. The role of oxidative stress in autoimmunity will be further established through iron overload, N-acetylcysteine administration and use of CYP2E1 knockout MRL +/+ mice; 2) Establish the role of RNS induced by TCE in autoimmunity. Using iNOS inhibitors and iNOS knockout MRL +/+ mice, we will evaluate the potential of RNS in leading to autoimmunity; 3) Elucidate the mechanism(s) of autoimmunity resulting from lipid peroxidation. Utilizing LPDA-protein adducts, the role of T cells in autoimmunity will be elucidated; 4) Define oxidative/nitrosative stress signature in the sera of SLE patients. Using sera from SLE patients, a link between oxidative stress markers and disease progression will be established. Our studies should establish oxidative stress as a pathogenic mechanism of ADs, and open important avenues for clinical intervention and medical surveillance through the development of disease markers, and will provide an avenue for mechanism-based risk assessment.
Relevance to Public Health: Autoimmune diseases are serious pathological conditions with unknown cause. Our goal is to elucidate the role of oxidative and nitrosative stress in the development of such diseases. The results of this study will provide important insight into the prevention and management of such diseases.
DESCRIPTION (provided by applicant): Our long-term goal is to elucidate the mechanism(s) by which environmental chemicals induce diseases so that preventive therapies and/or interventions can be devised. Trichloroethene (trichloroethylene, TCE), an environmental contaminant, is implicated in causing autoimmune-like diseases. However, no systematic studies have yet established its potential in inducing/exacerbating such diseases, or elucidated the mechanism(s) to TCE-induced autoimmunity, which is the focus of this application. Based upon the literature and our preliminary studies, we hypothesize that TCE and/or its metabolites bind to endogenous protein(s). These protein adducts elicit T and B cell responses that escape the normal tolerance to self-protein(s), leading to autoimmune-like diseases. This hypothesis will be tested by pursuing four Specific Aims: 1) To determine the autoimmune potential of TCE by conducting dose-and time-dependent studies in female MRL+/+ mice; autoimmune responses will be assessed by measuring autoantibodies and TCE-specific antibodies, circulating immune complexes, alterations in T cell functions, and by examining morphological changes. 2) To address the contribution of TCE metabolites to the induced autoimmunity, autoimmune responses to TCE-metabolites will be examined. 3) To identify the protein adduct(s) [antigen(s)] of TCE responsible for autoimmune responses. Protein adduct(s) will be purified from major tissues and characterized, and their autoimmune potential will be tested in mice. 4) To examine the role of T cells in TCE-induced autoimmune response. T cell responses (e.g., cell proliferation and the production of cytokines) to TCE and its protein-adducts(s) will be assessed. These studies should establish that TCE induces autoimmunity and provide insights into the mechanism(s) responsible for these responses. Our results will be important in designing strategies to prevent or reduce autoimmune responses to TCE and other related industrial chemicals. Furthermore, elucidating the mechanism(s) of TCE-induced autoimmunity will open avenues for early detection, medical interventions or therapies to prevent or manage various chemical-induced autoimmune diseases.
Crisp Terms/Key Words: environmental exposure, cell proliferation, autoimmunity, protein purification, electron microscopy, autoantibody, immunotoxicity, immune complex, immunofluorescence technique, enzyme linked immunosorbent assay, leukocyte activation /transformation, trichloroethylene, immunoglobulin M, immunoglobulin G, immunoglobulin A, systemic lupus erythematosus, adduct, cell differentiation, helper T lymphocyte, spleen, lymph node, laboratory mouse
DESCRIPTION (PROVIDED BY APPLICANT): Liver homeostasis is achieved by the removal of diseased and damages hepatocytes and their coordinated replacement to maintain a constant liver cell mass. Cirrhosis, viral hepatitis and toxic drug effects can all trigger apoptosis in the liver as a means to remove the unwanted cells, and the Fas 'death receptor' pathway comprises a major physiological mechanism by which this is occurs. The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor known to regulate both apoptotic and proliferative processes, and the AhR ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), is the prototype for a class of compounds known to affect these processes. Our long term goal is to understand mechanistically how the AhR contributes to tissue homeostasis by regulating cell growth and cell death. Our hypothesis, supported by the preliminary evidence, suggests that AhR activity sensitizes liver cells to Fas ligand (FasL) induced apoptosis, possibly by regulating expression of proteins that promote the cell death program. A plausible candidate is the AhR-regulated enzyme N-myristoyltransferase 2 (NMT2), because N-myristoylation of the Bid protein is critical for its activity in promoting FasL-induced apoptosis. The goal of this proposal is to study AhR function in the context of Fas-mediated liver apoptosis in vitro and in vivo. Aim 1 will examine whether the heightened susceptibility to Fas-mediated apoptotis depends on classical transcriptional activity by the AhR, or involves a non-classical mechanism. These studies will examine the severity of FasL-induced apoptosis in AhR-negative BP8 hepatoma cells expressing AhR molecules with targeted mutations that specifically disrupt AhR transcriptional activity. In Aim 2 we will determine whether the AhR-dependent susceptibility of hepatocytes to Fas-mediated apoptosis is due entirely to NMT2 action facilitating Bid activity. Aim 3 will examine the AhR's role in Fas-mediated apoptosis in isolated primary hepatocytes and in the liver in vivo. The studies will use an adenovirus gene transfer strategy to either express proteins, or use small interfering RNAs to suppress target gene expression in both cultured hepatic cells and the liver in vivo, in order to gain a mechanistic understanding of the functional relationship between the AhR and Fas-mediated hepatocyte apoptosis.
Crisp Terms/Key Words: small interfering RNA, protein protein interaction, CD95 molecule, green fluorescent protein, aromatic hydrocarbon receptor, gel mobility shift assay, enzyme activity, apoptosis, myristate, tissue /cell culture, protein structure function, fluorescence microscopy, HTC cell, liver function, homeostasis, transcription factor, gene expression, transfection, fatty acylation, polymerase chain reaction, flow cytometry, cell growth regulation, genetically modified animal, laboratory mouse
DESCRIPTION: Although smoking and alcohol use are major risk factors for squamous cell carcinoma of the head and neck (SCCHN), only a fraction of smokers develops SCCHN, suggesting a role for genetic susceptibility. We have found an association between increasing risk of SCCHN and suboptimal DNA repair capacity (DRC) that may be determined by adverse genetic variants such as single nucleotide polymorphisms (SNPs) in the nucleotide-excision repair (NER) genes. Therefore, we propose to focus in depth on the NER pathway by expanding from previously studied 5 non-synonymous SNPs (nsSNPs) to 85 common tagging SNPs (tSNPs) in the well-defined 8 core NER genes (i.e., ERCC1, XPA, XPB, XPC, XPD, XPE, XPF, and XPG), to correlate the variant alleles/genotypes or haplotypes/diplotypes with three NER phenotypes (i.e., NER mRNA expression, NER protein expression and NER DRC), and to evaluate their associations with risk of SCCHN. To accomplish these goals, we will include 1,600 SCCHN cases and 1,600 age-, sex-, and ethnicity-matched controls that consist of previously recruited 800 cases and 800 controls and an additional 800 cases and 800 controls to be recruited in this application using the same study design, selection criteria, and data collection instruments. To perform a comprehensive analysis of NER in SCCHN, our specific aims are: AIM 1: To determine the associations of the frequencies of variant alleles/haplotypes and genotypes/diplotypes of tSNPs in the 8 NER genes and the DRC phenotype with risk of SCCHN in 1,600 SCCHN cases and 1,600 controls. We will test the hypotheses that adverse alleles/haplotypes or genotypes/diplotypes of these selected genes and suboptimal DRC phenotype are associated with increased risk of SCCHN. AIM 2: To determine mRNA and protein expression levels of the 8 NER proteins by real-time RT-PCT assay and high-throughput reverse-phase protein lysate microarray assay, respectively, in cultured lymphocytes from 400 cases and 400 controls to be accrued. We will test the hypothesis that lower levels of NER mRNA and protein expression are associated with increased risk of SCCHN. AIM 3: To determine functional relevance of selected variant alleles and haplotypes of tSNPs of the 8 NER genes by correlating the frequencies of variant alleles/ haplotypes and genotypes/diplotypes with expression levels of mRNA and proteins of the 8 NER genes and DRC phenotype. This study will allow us to develop risk assessment models that integrate all biomarkers tested and epidemiological covariates. The relatively large sample size in this renewal allows for stratification analysis by tumor sites (i.e., oral cavity, pharynx and larynx), various genotypes/diplotypes and their combined genotypes, and epidemiologic covariates as well as for assessment of possible gene-gene and gene-environment interactions. This study will contribute to our understanding of the role of NER in the etiology of SCCHN and may lead to possible targets for primary prevention. PUBLIC HEALTH SIGNIFICANCE: This proposed study is to investigate the roles of genetic factors of DNA repair capacity (DRC), as well as their interactions with tobacco and alcohol use, in the etiology of squamous cell carcinomas of the oral cavity, pharynx, and larynx (SCCHN), expanding our preliminary findings to a role of DRC and its genetic determinants (85 single nucleotide polymorphisms, SNPs) in 8 DNA repair genes in a large study of 1600 cases and 1600 controls and applying our newly developed assays for mRNA and protein expression to 400 cases and 400 controls. Therefore, this study will help understand correlations between DRC genotypes (SNPs) and phenotypes (expression of mRNA and proteins and DRC) and their roles in the etiology of SCCHN. The long-term goal of this study is to identify effective biomarkers that can be used to identify at-risk individuals who will be targeted for primary prevention of SCCHN in the general population.
DESCRIPTION (provided by applicant): Inhalation of combustion smoke causes mortality and morbidity with immediate and delayed neurological impairments in survivors. In the current environment with escalating threats of terrorism, chemical warfare and combat situations, the risk of severe exposure to combustion smoke has significantly increased. Thus, the expanding scope of the problem necessitates urgent development of therapies to reduce neuropathology and long terms needs for care and rehabilitation. The development of targeted neuroprotective strategies however, is hindered since the molecular mechanisms underlying smoke inhalation neurotoxicity are not well defined. To understand the progression of neurotoxic events triggered by smoke inhalation, we developed a combustion-smoke inhalation model in the conscious rat. Our preliminary data demonstrates that the rat brain transcriptome and mitochondrial proteome are significantly altered by inhalation of smoke. Transcriptome changes peak at 24 hours and subside within 7 days post smoke injury. Overall, changes indicate concomitant activation of injurious and protective processes, with marked upregulation of genes involved in stress, cell death and protein degradation. In addition, we detect formation of oxidative damage in nuclear and mitochondrial DMA, and a delayed loss of hippocampal neurons several weeks after inhalation of smoke. We hypothesize that smoke inhalation impairs the fidelity of mtDNA repair and replication and that resultant, compromised integrity of the mitochondrial genome leads to dysfunction and loss of hippocampal neurons. To test this hypothesis, we have planned experiments with the following Specific Aims: 1) To delineate the effects of smoke inhalation on the oxidative DMA damage repair process in the brain. 2) To characterize smoke inhalation-induced alterations of the mitochondrial proteome, and determine to what extent mitochondria-encoded proteins are regulated at the transcriptional level and reflect loss of mitochondrial genomic integrity. 3) To elucidate mechanisms by which hyperbaric oxygen affects targets of smoke inhalation in the brain. Since our model is designed to mimic a real life situation, hyperbaric oxygen therapy, often given to smoke victims, is examined with respect to potential for protection of specific molecular and cellular targets of smoke. The overall objective of our proposal is to identify neurotoxic mechanisms contributing to delayed neuropathology in survivors of smoke inhalation. Our approach takes advantage of the novel rat model of smoke inhalation and integrates genomic and proteomic approaches to identify molecular mechanisms involved in the initiation and progression of smoke inhalation neurotoxicity, with the goal to establish a foundation for targeted neuroprotective therapies.
Crisp Terms/Key Words: neurotoxicology, neuroprotectant, combination therapy, oxidative stress, nonhuman therapy evaluation, hyperbaric oxygen therapy, longitudinal animal study, deoxyribonucleoside triphosphate, DNA repair, DNA replication, mitochondrial DNA, gene expression, pyruvate, pathologic process, molecular dynamics, hippocampus, brain, laboratory rat, air pollution
DESCRIPTION (provided by applicant): Unraveling the structural secrets of neuronal nitric oxide synthase (nNOS) has become an important goal for the purpose of understanding how nNOS can be differentially regulated and/or modulated by specific chemicals. The interaction of nNOS with environmental pollutants such as nitroarenes, resulting in the production of reactive intermediates and toxicity, is the subject of this proposal. In order to investigate the mechanisms of 1,3-dinitrobenzene (1,3-DNB)-mediated neurotoxicity, we hypothesize that in the presence of nitroarenes, nNOS is converted from a purely nitric oxide (NO*) and L-citrulline synthase to a peroxynitrite (ONOO-) and L-citrulline synthase. ONOO- is a very potent and reactive oxidant formed when nNOS simultaneously produces NO* and superoxide anion radical (O2-). O2- is formed by the nNOS-mediated reduction and subsequent reoxidation of nitroarenes. Concomitantly, nNOS maintains adequate electron flow to the heme to produce its normal products, NO* and L-citrulline. The simultaneous production of both NO* and O2- in close proximity leads immediately to ONOO- formation via the combination of these two radicals at a near diffusion-controlled reaction rate. The ONOO- that is produced, along with partially-reduced intermediates of the nitroarene, are proposed to play a role in the neurotoxicity associated with exposure to 1,3-DNB. The long-term objective of this project is to determine how metabolism of nitroarenes, resulting in the production of reactive intermediates (such as ONOO-, O2-, H2O2, NOx), and active reduced metabolites, mediate toxicity within the central nervous system. Further, we will determine how enzymatic activity of nNOS can be regulated by nitroarenes, O2-, and active reduced metabolites of nitroarenes such as the nitroso- and N-hydroxy-species. Toward this goal, our immediate specific aims are:
Aim #1: To dissect electron transfer from nNOS to nitroarenes such as 1,3-DNB by using recombinantly-expressed and purified nNOS and nNOS constructs.
Aim #2: To test the hypothesis that interaction of NOS with neurotoxic nitroarenes, including 1,3- DNB, results in modulation of NOS activity, stimulation of O2- production, and a gain of function by becoming a ONOO-generating enzyme.
DESCRIPTION (provided by applicant): Lead is a pervasive and potent neurotoxicant that produces persistent, concentration-dependent retinal, visual-motor, auditory and cognitive deficits in man and animals following exposure during development and adulthood. Approximately 2 million young children in the USA have blood [Pb] equal to or >10 mu g/dL, the currently accepted "safe" level, and millions more have levels of 2.5-10 mu g/dL, which place them at risk for these adverse health effects. Postnatal blood [Pb] equal to or >20 mu g/dL produce SUBNORMAL rod-mediated electroretinograms (ERGs). In contrast, recent results reveal that 7-10 year old children with low-level (blood [Pb] from 4-14 mu g/dL) gestational and continuous postnatal lead exposure have unique SUPERNORMAL rodmediated ERGs characterized by increases in a-wave amplitude, b-wave amplitude and sensitivity.
The overall objective of this research is to determine the sites and molecular mechanisms underlying ERG supernormality in children exposed to low-level lead during gestation. We developed a new rat model of lowlevel gestational lead exposure (blood [Pb] of 8-12 mu g/dL) that produces similar persistent supernormal rodmediated ERGs in adult rats. The proposed studies are designed to test the hypothesis that lead exposure during perinatal development produces ERG supernormality by altering the primary mechanism underlying the rod photoreceptor a-wave rod cGMP hydrolysis - and by altering the dopaminergic-modulated input underlying the b-wave amplitude and sensitivity. Specifically, we will determine whether perinatal lead exposure: 1) causes persistent supernormal ERG a- and b-waves by independent changes in rods and inner retinal neurons, respectively, 2) decreases the steady-state rate of rod cGMP hydrolysis by inhibiting the binding of any of the critical transcription factors to the rod cGMP phosphodiesterase beta-subunit promoter, and 3) produces TNF-alpha-mediated apoptotic cell death and dysfunction of dopaminergic retinal neurons resulting from the elevated retinal TNF-alpha levels measured following low-level perinatal lead exposure in rats.
The results from these functional (ERG), biochemical, molecular and immunocytochemical studies will: 1) determine the mechanisms underlying the rod-mediated ERG supernormality, 2) establish the critical period of retinal (neural) vulnerability during gestational development and 3) provide essential neurotoxicity data on low-levels of lead exposure that is of increasing scientific and regulatory concern.
Crisp Terms/Key Words: environmental exposure, neurotoxicology, gel mobility shift assay, cyclic GMP, prenatal stress, perinatal, embryo /fetus toxicology, high performance liquid chromatography, phosphodiesterase, lead poisoning, western blotting, enzyme linked immunosorbent assay, immunocytochemistry, tumor necrosis factor alpha, transcription factor, electroretinography, rod cell, retinal ganglion, hydrolysis, laboratory rat
DESCRIPTION (provided by principal investigator):
Background Toxin-mediated oxidative stress (OS) is a trigger that ultimately commits neurons to apoptotic death. The event can be associated with decreased cell glutathione (GSH) and cell death is mitigated by augmenting neuron GSH. Astrocytes protect neurons from toxin-related OS and subsequent apoptosis via the ?-glutamyl cycle, which maintains neuron GSH homeostasis. An understanding of this neuroprotective pathway, especially its regulatory components and species comparisons, could ultimately lead to novel clinical interventions for devastating neurodegenerative disorders as well as establish accurate model systems. Hypothesis: We hypothesize that within the astrocyte-neuron axis, there is a highly effective, regulated pathway consisting of components which can enhance neuron GSH homeostasis in response to EtOH (E) and Parkinson's Disease-producing environmental toxins, thereby minimizing death of neurons. However, there are exposure patterns to these environmental cross-stressors which damage components of the pathways, thereby impairing its neuroprotective capacity. Specific Aim 1: Aim one will utilize cultures of neurons and astrocytes, alone and in co-culture, to address the direct impact of rotenone, paraquat, and E on astrocyte and neuron components of the ?-glutamyl cycle. Experimental parameters will be effects of the environmental toxins, alone or in combination, on inward transport of key precursors of GSH, GSH enzymatic synthesis, and components of this machinery at which neuroprotection occurs. Specific Aim 2: Aim two will address regulation of three essential components of the neuroprotective pathway. To be determined will be basic mechanisms underlying regulatory and damaging responses to the toxins. These will be regulation of GSH efflux via multidrug resistance protein(s) (Mrp), and two plasma membrane ectopeptidases, ?-glutamyl transpeptidase (GGT) and aminopeptidase N (ApN). Specific Aim 3: Aim 3 will use an in vivo model to extend the in vitro findings to the intact animal. Experiments will utilize two-photon excitation microscopy to determine toxin effects specifically in cortical astrocytes and neurons in the living brain. These will be time-lapse determinations of expressions of GGT and ApN, GSH content and apoptosis-related events during toxin exposure. They will utilize transgenic mice deficient in Mrpl, Mrp4, or GGT. General Description: The proposal addresses a new system by which glial cells protect neurons from the toxic effects of three environmental toxins. These compounds can cause neurons to die as a result of oxidative damage and cells called astrocytes prevent this by maintaining neuron antioxidants. We will determine how the toxins damage it (to prevent this from happening), and how this system is normally controlled.
DESCRIPTION (provided by applicant): Inappropriate exposure to environmental agents during critical, sensitive periods of development can reprogram target tissues via epigenetic mechanisms to increase the risk for disease later in adult life. The reproductive tract is particularly sensitive to this type of developmental programming in response to xenoestrogen exposure, a major class of endocrine disrupting chemicals that are ubiquitously present in our environment. We have recently shown that developmental programming following neonatal xenoestrogen exposure can increase the risk of developing hormone-dependent leiomyoma by increasing the responsiveness to hormone of estrogen-responsive genes in the myometrium. Although the epigenetic mechanism(s) responsible for developmental programming are unknown, recent data indicate that one type of epigenetic modification, histone methylation, is altered in response to activation of PI3K signaling via phosphorylation of histone methyltransferases by AKT. Since estrogens can activate PI3K via nongenomic signaling, we hypothesize that xenoestrogens, via activation of cell signaling pathways, can modify the activity of histone methyltransferases, leading to epigenetic alterations in histone methylation patterns that contribute to developmental programming in the uterus. To test this hypothesis, we propose the following Specific Aims: Specific Aim 1: Test the hypothesis that estrogen-activation of cell signaling results in changes in histone modifications that alter gene expression. Specific Aim 2: Determine if phosphorylation of methyltransferases by AKT or ERK is responsible for changes in histone modifications that occur in response to estrogens. Specific Aim 3: Determine the contribution of histone modifications to developmental programming of gene expression in the uterus. These studies will explore activation of cell signaling as a novel mechanism by which histone methyltransferase activity can be modulated by estrogens, resulting in epigenetic alterations in histone methylation patterns. In addition, they will determine if changes in histone methylation induced by neonatal exposure to xenoestrogens are heritable and contribute to developmental programming by modulating gene expression in adult animals.
PUBLIC HEALTH RELEVANCE: Uterine leiomyoma are the most common gynecologic tumor of women and they have a tremendous negative effect on the health of millions of women in the US. Surprisingly, we know very little about what causes these tumors, although hormones, including environmental estrogens, have been implicated in the etiology of this disease. We propose that a potential mechanism by which environmental estrogens may contribute to the development of these tumors is through developmental programming. In this application we will test the hypothesis that during development of the uterus, exposure to environmental estrogens reprograms gene expression in the myometrium to predispose to the development of these tumors later in life. The mechanism that we propose for this reprogramming is the activation of nongenomic signaling pathways to modulate the activity of histone methyltransferases and induce alterations in epigenetic patterns of histone methylation.
DESCRIPTION (provided by applicant): Manganese (Mn) is a natural element that can be both beneficial and toxic. The long-term goal of this study is to define the effects of Mn exposure on the female pubertal process. Our recent studies are the first to show that Mn can stimulate prepubertal luteinizing hormone (LH) secretion by an action at the hypothalamic level, prompting us to question whether Mn is involved in the pubertal process and whether chronic oral exposure to low but elevated levels of Mn at an early age might advance female puberty. Our preliminary evidence indicates that it can advance puberty an action associated with elevated levels of important puberty related hormones. This is a novel and potentially important observation since precocious puberty is a major child health concern and in young girls the cause is often unknown. We hypothesize that if Mn levels rise too early in life, the seemingly beneficial effect of Mn to facilitate LH release may actually cause or contribute to female precocious puberty. The following specific aims will be used to critically address this important health related concern: AIM 1) Determine the minimum effective dose and critical time of Mn exposure to elicit precocious development. AIM 2) Assess effects of Mn on hypothalamic genes associated with control of gonadotropin secretion and correlate changes with specific puberty related hormones and receptors. AIM 3) Conduct hypothalamic and pituitary response tests to demonstrate the Mn influence on precocious puberty is of central origin. AIM 4) Assess pharmacologically the specific receptors and post-receptor pathways involved in the hypothalamic LH-releasing hormone (LHRH) pathway of secretion to determine the site(s) of Mn action. AIM 5) Begin assessing mechanism(s) by which Mn facilitates LHRH release. The proposed studies are needed to define the actions and interactions of Mn on female pubertal development and are clearly relevant to child health and well being.
DESCRIPTION (provided by applicant): Project Summary: The objective of this study is to elucidate the mechanism by which testosterone inhibits spermatogonial differentiation after toxicant exposure of rats. Radiation will be used as a model toxicant in most studies and the relevance to chemical toxicants will be assessed by comparison with results obtained after dibromochloropropane (DBCP) exposure. In particular, the extension to DBCP of the conclusion that radiation induces the spermatogonial differentiation block by damaging the somatic environment, not the spermatogonia, will be examined. In irradiated rats, the source of the factors and the specific factors that are regulated by testosterone to affect spermatogonial differentiation will be identified. Previous results indicated that factors present or transmitted through the interstitium are important. That hypothesis will be tested by examining spermatogonial differentiation in vivo after testicular somatic cell or tubule transplantations or after selective depletion of the interstitial Leydig cells or macrophages, which produce paracrine factors, and by assessing the effects of interstitial fluid on spermatogonial differentiation in vitro. Next, genes coding for growth and differentiation factors that are regulated by testosterone and FSH in a manner coordinate with spermatogonial differentiation will be further tested for their correlation with spermatogonial differentiation using other hormonal modulations; the gene that best correlate will be studied, along with their receptors, in depth. An in vitro system will be used to determine whether testosterone or other hormones directly modulate spermatogonial differentiation independent of systemic effects that occur in vivo and also to test whether the specific protein factors regulated by testosterone affect spermatogonial differentiation. Relevance: An increasing number of men have low sperm counts, possibly due to exposure to known and unknown environmental agents. We have developed a model in which radiation and chemical toxicants produce prolonged reduction or absence of sperm in rodents, despite the presence of spermatogonial stem cells. We have also developed hormonal methods for the reversal of this block in spermatogenesis; however the mechanisms of the block and its reversal are unknown. It is essential to elucidate these mechanisms in order to determine how methods for reversal of that block could be applied to man.
DESCRIPTION (provided by applicant): The prevalence of asthma and other allergic diseases in industrial countries have increased dramatically. Cells of the innate immune system (mast cells, basophils and eosinophils) contribute directly to the allergic inflammation and can promote the differentiation to the T helper type 2 (Th2) phenotype that supports isotype switching of B cell to IgE production and late phase responses. The initial priming of the T helper cell system to allergen frequently occurs in utero or in the early postnatal period, when Th2 cells normally dominate the immune response. Factors that delay the normal conversion from Th2 to Th1 dominance enhance the risk of developing allergic diseases. We have found that exposure to estradiol (E2) and environmental estrogens strongly potentiate the synthesis and release of allergic mediators from mast cells, through a membrane form of estrogen receptor alpha. Further, ovarectomized BALB/c mice that fail to make an IgE response after typical allergic sensitization are reconstituted by implanting E2 pellets. Environmental estrogens tend to degrade slowly, bioaccumulate and bioconcentrate in the food chain, and are transferred to offspring via the placenta and breast milk. The current proposal is to develop an animal model of asthma for identifying critical developmental period(s), during which environmental estrogens promote allergic sensitization and airway hyperreactivity and inflammation. We will also use this model to perform initial ex vivo experiments to identify the key cell type(s) that are functionally altered by environmental estrogens. The general hypothesis to be tested in this project is that exposure of genetically susceptible animals to environmental estrogens during critical developmental period promotes allergic sensitization / reactions by modulating the function of cells of innate immunity. The specific aims that will test the specific hypotheses that: 1) Exposure to environmental estrogens, during a critical period(s) of immune development can enhance allergic sensitization and reactions in an animal model of asthma, and 2) Environmental estrogens enhance allergic sensitization by modulating the potential of antigen presenting cells (APCs) to support Th2 responses, shaping the responses of T cells to normal APCs, and/or enhancing mast cell degranulation and production of cytokines. More extensive future studies will dissect the molecular mechanisms that can help us identify approaches to prevent exposures or interrupt adverse estrogenic effects. Environmental pollution is a likely contributor to the recent increase in asthma and other allergic diseases. This project will develop a mouse model to investigate the effects of environmental estrogen-like chemicals on the development of asthma in children.
DESCRIPTION (provided by applicant)
Polycyclic aromatic hydrocarbons (PAHs) constitute a complex group formed from incomplete combustion of organic carbon and are found in significant amounts in automobile exhaust, cigarette smoke, various foods, and industrial waste by-products. They represent serious, ubiquitously environmental contaminants. PAHs contamination of individuals occurs by inhalation, ingestion, or contact. Some PAHs are carcinogenic, teratogenic, and mutagenic. Biomonitoring is essential to make precise, quantitative measurements of personal exposure to environmental chemical/biological agents because it can provide an assessment of the integrated uptake through all exposure routes. Conventional biomonitoring methods are often complex, time-consuming, labor intensive, and expensive.
Immunoassay is probably the most commonly used technology for the detection and quantification of biomolecules in the diagnosis and management of disease. Current trends in the development of immunoassay methodologies are miniaturization, multiple detection, and automation. On the other hand, nanotechnology has recently boomed because of the availability of new investigative tools. This technology makes it possible to characterize chemical and mechanical properties at a single molecular level, discover novel phenomena and processes, and provide science with a wide range of tools, materials, devices, and systems with unique characteristics.
The aim of this project is to develop a novel multiple biological sensor combining immunoassay (antibodybased detection) and emerging nanotechnology (a nanoscale biosensor) to assess real time and continuous readout of internal exposure to priority environmental chemical agents, specifically to PAHs. In this innovative approach, the presence of targeted biomolecules in serum is monitored by specific antigen-antibody interaction at the nanoscale. Specific antigen-antibody interactions make this approach highly precise to identify specific antigens, such as biomarkers for PAH exposure, enabling accurate assessment of the exposure. Other advantages of this approach over conventional immunoassay techniques are (i) no other signal-generating moieties (labeling agents) are needed to measure the antigen-antibody interaction, (ii) multiplex assessment of informative and predictive serum biomarkers is possible, and (iii) wide range of applications. During the Phase I project, the proof-of-concept will be established, including the detection of antibody/antigen interactions in serum and in the presence of other biomarkers of other environmental exposure set.
The proposed system has a high potential for user acceptance and has immediate commercial applications in many areas. Key segments in the medical diagnostic markets where the proposed technology can have an immediate impact include hospitals, clinics, clinical laboratories, medical insurance companies, and point-of-care uses.
DESCRIPTION (provided by applicant)
Pesticides pose a significant health hazard to the general public and especially individuals working or living around farmland. Pesticides can enter the drinking water supply via run-off into lakes and streams. People can be exposed to pesticides by inhalation, swallowing or by skin or eye contact resulting in poisoning symptoms including: excessive sweating, chills or thirst, chest pains, difficulty breathing, and muscle cramps. To help prevent exposure to these toxic compounds, Lynntech proposes to develop a wearable/portable sensor that can detect, identify and determine the concentration of these compounds within a variety of matrices, i.e. air, water and/or soil in a rugged and reliable device. Using cutting-edge nanotechnology, Lynntech will fabricate miniaturized sensors coupled with innovative enzyme matrices that have the potential to identify P-O, P-S, and P-F type pesticides individually, providing an improved sensing platform for critiquing their source. The proposed sensor technology will be extremely lightweight and long-lasting due to its lowpower requirement, small size, and enhanced enzyme stabilization. The proposed pesticide sensor will have commercial applications in both remote and/or point-of-use monitoring all water supplies, protecting farmers/crop-sprayers or military soldiers in the battlefield against chemical warfare agents which are often in the form detectable by the proposed sensor.
Lynntech's Phase I research will consist of several proof-of-concept experiments which will involve the demonstration and characterization/optimization of the signal response's sensitivity, selectivity and propensity to give false-positive responses. Phase II research will continue with the advancement of the sensing platform with the design, assembly and testing of the first-generation wearable sensor with automated sampling, processing and data display. The research team consisting of a group of research engineers and scientists with backgrounds in sensor design and fabrication, enzyme characterization, software/hardware programming and electrochemistry, making this research project truly multidisciplinary, thereby providing the necessary expertise to make this project a success.
DESCRIPTION (provided by applicant): Recent scientific investigations have shown that many chemicals used in plastics, pharmaceuticals, pesticides, cosmetics, food additives, etc., are endocrine disruptors (EDs). EDs interfere in various ways with hormones such as estrogens to have significant adverse effects on many behavioral and physiological processes. ED effects (e.g. estrogenic or anti-estrogenic) sometimes occur at very low (picomolar to nanomolar) concentrations, especially on fetal or developing mammals (including humans). The prevalence and actions of EDs in our environment warrant the development of valid assay methods. Consequently, various governmental bodies (e.g., EPA, FDA, and ICCVAM) and proactive corporations have explicitly expressed a desire to have in vitro robotic assays for EDs, such as anti-estrogenic activity (anti-EA). However, no robotic anti-EA assay is commercially available. To begin to meet these governmental, scientific and commercial needs, CertiChem (CCi) has completed a Phase I SBIR grant showing that it is feasible to develop an anti- EA robotic assay that would be valid (i.e., reliable, accurate, versatile, rapid, and cost effective) using MCF-7 cells. In this Phase II application, CCi now proposes to develop this robotic assay for anti-EA for commercialization by repeatedly assaying a set of 78 reference test chemicals to confirm the reliability and accuracy of CCi's proposed anti- EA assay. CCi also proposes to demonstrate the versatility of this assay by examining the anti-EA in a set of antioxidants used in foodstuffs and plastics. Finally, CCi proposes to perform HPLC separation assays on some reference test chemicals to confirm that their anti-EA is not due to contamination and to enhance the versatility of this assay for commercial use. Development of a robotic screening assay for anti-EA is commercially, scientifically, and socially important because of the large number of chemicals (>10,000) - much less chemical mixtures - that should now be screened for anti-EA by profit, non-profit, or governmental entities. Development of a robotic assay for anti-EA is desired as part of the mission of NIEHS, EPA and ICCVAM. Recent scientific investigations have shown that many (perhaps over 10,000) chemicals used in common products such as plastics, pesticides, cosmetics, and food additives have estrogenic/anti-estrogenic activities that severely interfere with normal estrogen actions to produce adverse effects on many behavioral, reproductive, and physiological processes in humans. In this Phase II proposal, CertiChem (CCi) proposes to develop a very sensitive and accurate assay to measure anti-estrogenic activity in a set of reference test chemicals and in common consumer products. CCi proposes to use these data and CCi's data on estrogenic activity on many chemicals to identify which chemicals have anti-estrogenic activity and to work with various firms to design consumer products that are free of chemicals that release anti-estrogenic or estrogenic activities.
DESCRIPTION (provided by applicant): The availability of safe drinking water is a substantial health concern. The introduction of water chlorination as a standard water treatment has resulted in a significant decrease in the number of waterborne diseases. However disinfection byproducts (DBPs) are formed by the reactions between chlorine and the natural organic matter (NOM) dissolved in water. Long-term exposure to DBPs may increase the risk of cancer and other adverse health effect. As a consequence, the U.S. Environmental Protection Agency (EPA) has implemented stringent limits for DBPs in drinking water. Although the use of alternative disinfectants have been proposed, they also produce their own toxic byproducts. And water plant approaches to reduce the amount of NOM in the source water prior to chlorination step are not technically sound due to the requirement of a new water treatment plant that is costly to construct and operate. Thus, new methods for elimination of DBPs in drinking water at the point-of-use (POU) are strongly needed.
The objective of the Phase II proposal is to design, fabricate and test a new, small-scale photocatalytic oxidation system for POU water treatment. The aim is to provide a unique, low-cost consumer device designed to effectively eliminate DBPs and microorganisms at the location where water is consumed. The proposed system provides attractive benefits over other POU technologies because it adds nothing harmful to the water, does not require routine service, does not provide a harbor for microorganism growth, and water contaminants including DBPs and microorganisms are destroyed, not just transferred from water to solid phase, and therefore, it does not create a waste disposal problem.
During the Phase I project, Lynntech developed and successfully tested bench scale photocatalytic systems capable of fast degradation of a variety of microorganisms and DBPs to well bellow EPA recommended levels. It overcame limitations of other photocatalytic system including deactivation by inorganic ions. The heart of Lynntech's water treatment is the use of effective photocatalyst nanoparticles immobilized on an active matrix. The photocatalyst material is integrated into an ingenious reactor design that minimizes mass transfer and allows for fast treatment times. During the Phase II, an optimized automated GEN I POU photocatalytic water treatment will be developed. We will test it for its reliability, repeatability, ease of use, manufacturability and ease of assembly while minimizing its cost. Its operation will be on-line, and user interaction will be limited to the replacement of the UV lamps after about 5,000 hours of use. GEN I POU system will be extensively tested with DBPs and microorganisms commonly found in tap water.
The proposed system has a high potential for user acceptance and has immediate commercial applications in many areas where high quality water is needed. Key segments where the proposed technology can have an immediate impact include hospital, schools, pharmacies, cafeteria, commercial and residential buildings, etc.
The availability of safe drinking water is a substantial health concern. The introduction of water chlorination as a standard water treatment has resulted in a significant decrease in the number of waterborne diseases, but disinfection byproducts (DBPs) are formed by the reactions between chlorine and the natural organic matter (NOM) dissolved in water, and long-term exposure to DBPs may increase the risk of cancer and other adverse health effect. The aim of this Phase II proposal is to provide a unique, low-cost, small-scale photocatalytic oxidation system for point-of-use water treatment device designed to effectively eliminate DBPs and microorganisms at the location where water is consumed.
Crisp Terms/Key Words: nanotechnology, nonbiomedical equipment, technology /technique development, water treatment, water sampling /testing, microorganism toxicology, photochemistry, oxidation, catalyst
DESCRIPTION (provided by applicant): The goal of this SBIR project is the development of an improved inorganic ion exchanger with an extremely high selectivity for Sr2+. This ion exchanger will improve the extraction of 90Sr from groundwater at the sites around the US and the world that have become contaminated through mishap or poor handling of nuclear materials, some of which are in contact with aquifers and bodies of water used as drinking water supplies. 90Sr is a ?-emitting isotope with an extremely low maximum safe exposure limit. (The drinking water limit is 8 pCi/L.) Development of this material will make a significant contribution to safer drinking water. In Phase I of this project we demonstrated that the substitution of SnlV and SilV for part of the SbV in the pyrochlore-structured antimonic acid (Sb2O5 4 H2O) increased both the compound's affinity for Sr+2 and its selectivity for strontium over calcium by a factor of nearly four. (A strong preference for Sr+2 is important, because the strontium is always found with a large excess of calcium.) The resulting material has a capacity for 90Sr over 200 times that of the clinoptilotile currently used to collect strontium in pump-and-treat systems. In Phase II we will refine the composition of the exchanger and scale-up the synthesis. The material will be evaluated in complete detail for use as a column material for extracting 90Sr from ground water out of the ground or as the active component in a permeable reactive barrier to block the motion of 90Sr in ground water in the ground. In plain language: Adding tin or silicon to antimonic acid greatly improves its ability to remove strontium from aqueous solutions in the presence of the very similar calcium. This makes it an excellent choice for removing radioactive strontium-90 from ground water either at the surface, or while still in the ground.
Crisp Terms/Key Words: chemical synthesis, strontium, tin, antimony, ion exchange chromatography, radionuclide, radioactive waste, silicon, environmental toxicology, ground water, water pollution, water sampling /testing, water treatment, technology /technique development, calcium ion
DESCRIPTION (provided by applicant):
Bitter-taste perception is a key mechanism of environmental response in humans. By allowing the individual to recognize potentially toxic compounds, bitter-taste perception allows control over their intake. Historically, the ability to perceive bitter compounds likely provided evolutionary advantages by allowing humans to avoid poisoning by plants, many of which use bitter toxins as a defense against herbivores. Many well-known bitter compounds such as quinine, nicotine, and caffeine, for example, are toxins produced naturally by plants. In modern populations, bitter-taste perception continues to be an important determinant of health through its effects on diet choice and other taste-related behaviors. For instance, significant associations are found between sensitivity to bitter compounds such as phenylthiocarbamide (PTC) and 6-n-propylthiouracil (PROP) and major health factors such as body mass index (DMI) and smoking tendencies. Given the importance of bitter-taste in human evolution and health, the applicant would like to know how evolutionary pressures have shaped patterns of variation in the genes underlying bitter-taste perception, and how these patterns of variation in turn shape bitter-taste phenotypes. This project will address patterns of genetic variation in human bitter-taste receptor genes to investigate the roles of demography and natural selection in shaping variation in these genes, and the contribution of variation in these genes to variance in bitter-taste phenotypes. First, 24 bitter-taste receptor genes will be resequenced in an ethnically diverse sample of 190 humans from 14 populations in Africa, Asia, Europe, and the Americas. Next, the data will be analyzed using population genetic methods to determine the roles of demography and natural selection in shaping patterns of diversity in each gene. Information about these effects will then be used to identify polymorphisms that have likely been maintained by balancing selection and local adaptation. Finally, variance component-based linkage analyses will be applied to the candidate polymorphisms and all polymorphisms to determine the relationship between variation in bitter-taste receptor genes and three phenotypes: PTC sensitivity, quinine sensitivity, and DMI. By applying a combined evolutionary and epidemiological approach, these analyses may shed new light on the relationship between genetic variation, environmental response, and human health.
DESCRIPTION (provided by applicant)
The candidate's research career began by gaining an understanding of the fundamentals of recombination, in particular the genetics of homologous recombination (HR). Initially, as a graduate student, the candidate used the well-understood genetic system Saccharomyces cerevisiae. The candidate then moved on to work with a mouse model system during postdoctoral studies, being successful in establishing a novel assay system that allowed the determination that both the endogenous level of HR and the carcinogen induced HR were dependent upon the damage response pathway. Information and experience gained from these studies directed the candidate to the hypothesis that there are other cellular components that affect genomic stability and will also play a part in cellular damage response. These ideas can be pursued using state-of-the-art RNA interference (RNAi) technology, an available Drosophila genome library of RNAi and Drosophila tissue culture. This study will provide the resources, tools and data required to eventually establish an independent research group and launch an independent academic career. As a longer-term goal, the candidate hopes to establish a novel in vivo recombination assay and utilize it to examine both the genes identified in this proposal and other well established determinants (genetic and pharmacological) of genomic stability in an in vivo and tissue specific manner. Information gained from these studies, together with the novel tools established, should open up a wealth of research opportunities. This project has been made possible by the candidate's present position and mentor Dr. Philip Leder, who has encouraged the development of independent ideas and has taken a strong interest in this project. In addition to the highly supportive research group, the department provides an interactive community that fosters many collaborative opportunities. The objective of this project is to determine as yet unidentified components of the cellular damage response system that are responsible for either maintaining a basal frequency of HR in the presence of endogenous damage or the induction of HR following exposure to an exogenous agent. The specific aims are to: 1) develop and conduct a genetic screen for modifiers of HR in Drosophila using an available RNAi library and 2) determine whether mammalian homologues of two or three identified Drosophila genes act in an analogous fashion. From this work I hope to identify the genes that will form the basis of my future work.
DESCRIPTION (provided by applicant)
Environmental Toxicology training at Texas A&M University is coordinated
through the Interdisciplinary Faculty of Toxicology (IFT) which has
faculty membership from all the major Life Sciences Departments. Preand
postdoctoral trainees also participate in the multiple health
science initiatives including the Center for Environmental and Rural
Health (CERH), Superfund Basic Research Program (SBRP) and the
Partnership for Environmental Education and Rural Health. All trainees
will complete core course requirements in toxicology and satisfy IFT,
departmental, and University academic requirements. Trainees will also
participate in multiple enrichment activities which include: (a)
participation in annual research symposia sponsored by IFT and CERH;
(b) attendance at weekly [FT seminars; (c) participation at both
regional (Gulf Coast) and national Society of Toxicology meetings; and
(d) participation/attendance at other local, regional and national
scientific conferences. Special emphasis on minority recruitment will
continue to be an important objective of this grant. The proposed
training program will provide a challenging research program focused on
diverse areas of environmental toxicology that will be directed by ten
co-investigators. Research programs of training faculty include:
fluorescent/imaging analysis of toxicant and hormonally-induced injury
(Burghardt); bioassay-directed analysis of endocrine-active chemicals
and marine mammal genetics (Busbee); risk assessment and genotoxic
mechanisms of PAH mixtures (Donnelly); gene-environment interactions and
their modulation of embryonic development (Finnell); male reproduction
and spermatogenesis (Johnson); development of clay-based technologies
for toxicant-specific adsorption (Phillips); genetic and epigenetic
modulation of vascular and renal cell proliferation and differentiation
(Ramos); mechanisms of estrogen receptor-mediated gene expression and
inhibitory Ah receptor crosstalk in breast cancer (Safe); mechanisms of
heavy metal-induced neurotoxicity in cell culture and in vivo (Tiffany-
Castiglioni); mechanisms and applications of organophosphorus degrading
genes (Wild). The proposed training program will continue to provide
trainees with a challenging academic and research environment that will
prepare them for future careers in academic, industry and government.
DESCRIPTION (provided by applicant): Exposure to environmental chemicals during specific, susceptible periods of fetal development can reprogram target tissues and increase disease incidence later in life. Xenoestrogens are a class of endocrine disrupters found throughout our environment that are capable of reprogramming a number of tissues, including the female reproductive tract. Previous studies from our laboratory demonstrated that perinatal xenoestrogen exposure reprograms estrogen-responsive genes in the myometrium and elevates leiomyoma incidence in rats. The mechanisms underlying reprogramming and subsequent tumorigenesis in uterine tissue following xenoestrogen exposure are unknown. However, interaction between estrogenic signaling and epigenetic regulation of gene expression such as changes in histone and DMA methylation is the probable cause of this developmental programming. Recent studies indicate that histone methylation is a precursor for the establishment of permanent epigenetic states within the genome. In addition, exciting new data demonstrate that cell signaling pathways, which can be activated by estrogen via non-genomic signaling, modulate the activity of histone methyltransferase (HMT) enzymes. Based on this information we hypothesize that xenoestrogens alter chromatin structure via aberrant modulation of histone methylation, causing permanent reprogramming of genes. To address this hypothesis, we propose the following Specific Aims: Specific Aim 1) Determine if estrogen receptor signaling alters HMT activity. Specific Aim 2) Determine if estrogens induce gene-specific changes in histone methylation. Specific Aim 3) Determine if the ability to alter HMT activity and induce gene-specific methyl marks is shared across broad classes of xenoestrogens.
PUBLIC HEALTH RELEVANCE: The proposed studies will be used to carefully dissect the impact of estrogen signaling on HMTs using chromatin immunoprecipitation, HMT activity assays, transfection of constitutively active and dominant negative signaling proteins, and site-directed mutagenesis. The goal of this application is to investigate a novel mechanism of action for environmental estrogens.
DESCRIPTION (provided by applicant)
This a competing renewal application of an environmental toxicology training grant for 6 pre-doctoral fellows and 3 postdoctoral fellows. Since this program started in 1990, training has been provided to 41 pre-doctoral fellows and 18 postdoctoral fellows including 5 physician-scientists. The 39 trainees who have completed their research training have professional positions where they utilize their toxicology knowledge for basic research, biotechnology, teaching, risk assessment or practical problems of environmental pollutant regulation and policy. Ten have academic faculty appointments. Three who subsequently obtained law degrees are practicing environmental or patent law. The long term goals of those who are pursuing further training in academic postdoctoral fellowships are basic research, teaching, science policy, or translational medicine. A tangible outcome of this policy about trainee submission of competitive research proposals is that 27 have obtained individual fellowships, including 11 from NIEHS. The propose research training in three areas where the investigators have a critical mass of experienced faculty, exemplary institutional support, and superb resources for state-of-the-art research, notably for proteomics. These areas are biotransformation, DNA damage and repair, and pathophysiology which share a unifying theme of oxidative stress. Training in the broad area of pathophysiology will emphasize injury to the CNS (brain and eye), GI tract, and asthma pathogenesis which meshes with the strong participation by physician scientists in the Asthma Pathogenesis Research Core of the newly renewed NIEHS Center Grant. A unique feature of the proposed training is that research projects by several faculty members in each area include assessments of the influences of aging and infection. An updated curriculum builds from an interdisciplinary common first year towards advanced courses in molecular toxicology, pathology, proteomics/bioinformatics, and intensive short courses on specialized topics that are taught by visiting scientists. Identity with the interdisciplinary program is established by a regular dinner-journal club and seminars. Professional development is facilitated by required participation in a team-taught undergraduate toxicology course which is organized, taught, and graded by trainees with faculty guidance. Key elements in the investigators' outstanding climate for training are a multiplicity of environmental-health relevant research centers, notably the recently renewed Institutional Sealy Center for Environmental Health and Medicine which will provide $50,000/year for training program recruitment and pilot projects for trainees.
DESCRIPTION (provided by applicant): Recent studies demonstrate that the aryl hydrocarbon receptor (AhR), a transcription factor, plays an active role in liver homeostasis, and that this process is dysregulated by the environmental toxicant 2,3,7,8- tetrachlorodibenzo-p-dioxin (TCDD). TCDD is a potent AhR agonist responsible for activation and nuclear translocation of AhR from the cytosolic compartment. Previous studies determined that AhR-mediated transcriptional activity requires partnering with the nuclear ARNT protein and DNA binding of this heterodimer to a consensus xenobiotic response element (XRE) linked to target genes. Preliminary evidence examining TCDD induction of plasminogen activator inhibitor-1 (PAI-1), a key protein in liver regeneration, exposed a novel AhR-dependent regulatory mechanism involving DNA binding to a nonconsensus XRE (NC-XRE) independent of the ARNT protein. We hypothesize that the AhR regulates PAI- 1 gene expression by a formerly unrecognized mechanism of action. This study will identify the components of the complex bound to the NC-XRE and characterize the AhR protein-protein and protein- DNA interactions at this site. Specific Aim 1 will employ DNA-affinity chromatography using the NC-XRE binding site characterized by EMSA and functional studies to fractionate the complex from mouse liver nuclear extracts following induction with TCDD. MALDI-TOF/TOF will be used to identify the proteins in the NC-XRE-bound complex. Specific Aim 2 will verify immunologically that the proteins identified in aim 1 are indeed components of the NC-XRE binding complex using EMSA supershift assays and coimmunoprecipitation studies. Studies in Specific Aim 3 will focus on characterizing the nature of AhR interaction in this complex through a series of in vitro assays designed to examine protein-protein and protein-DNA interactions. Collectively, these studies will explore a new mechanism in the growing repertoire of distinct AhR activities that must be resolved if biologists are to fully understand the physiological role of the receptor or obtain reliable human health risk assessments following exposure to TCDD and related environmental toxicants.
DESCRIPTION (provided by applicant) The investigators are requesting the continuation of a training grant in support of a predoctoral program promoting excellence in the toxicological sciences, focusing on molecular mechanisms of environmental chemical induced toxicity and disease. The Program is located at two geographically close sites within the University of Texas (UT) system, and which have multiple, well established training and research collaborations. These interactions are fostered by a joint NIEHS sponsored Center for Research on Environmental Disease (CRED) and a shared T35 training grant that supports summer research training for undergraduate minority students on both campuses. An important aspect of this program is the graduation of trainees who are actively recruited by colleagues from around the nation. This training program is particularly effective at attracting underrepresented groups into the discipline of Toxicology (women and minorities). Although each trainee selects from a wide variety of ongoing research programs, and works primarily in the laboratory of one of the training faculty, the training program promotes and supports collaborative research. The research programs of the training faculty include: 1) mechanisms of experimental and human carcinogenesis (Conti); 2) mechanisms and regulation of protein kinases (Dalby); 3) molecular mechanisms of multi-stage skin carcinogenesis (DiGiovanni); 4) role of inflammation in chemical carcinogenesis (Fischer); 5) hormonal carcinogenesis (Fuchs-Young); 6) E2F transcription factors (Johnson); 7) free radical biochemistry and molecular mechanisms of apoptosis (Kehrer); 8) role of vitamin E in signaling pathways leading to cell death (Kline and Sanders); 9) metabolism, chemical-induced nephro-carcinogenicity, prostaglandin mediated cytoprotection and proteomics (Lau); 10) molecular interactions of carcinogens with chromatin (MacLeod); 11) molecular mechanisms of oncotic and apoptotic cell death in response to reactive oxygen species (ROS)-induced DNA damage, ROS-induced changes in chromatin structure and function (Monks); 12) male reproductive toxicology and germ cellapoptosis (Richburg); 13) mechanisms of apoptosis in epithelial tumorigenesis (Tang); 14) mechanisms of DNA damage recognition by DNA repair and recombination pathways (Vasquez); and, 15) role of genetic alterations in tumor development (Walker). Predoctoral trainees are evaluated for admission into the training program on the basis of GPA, GRE, letters of recommendation previous research experience, and interviews. Progress is monitored throughout the year by participation, each semester, in seminars, course work, research progress in the laboratory, and an annual report prepared by the trainees describing their progress. Faculty have an excellent history of collaboration and sharing of research resources, such as advanced instrumentation, that foster multidisciplinary research. Thus, (i) the maturity of the graduate program, (ii) the clear demand for the graduates which exceeds the investigators' supply, (iii) the institutional commitment to the educational mission of the toxicology program via the establishment of the Center for Molecular and Cellular Toxicology, (iv) in combination with the established NIEHS Center (CRED), all combine to provide an environment ideal for an increase in the number of NIEHS supported predoctoral positions. The investigators therefore believe this is the optimum time to expand the training grant.
DESCRIPTION (provided by applicant)
This proposed STEER program is intended to support the annual participation of eight academically talented undergraduates in the Environmental Health Sciences Summer Undergraduate Research Program (EHS-SURP). The overall goal of EHS-SURP is to provide an authentic, hands-on, ten-week bench research experience for students considering careers in EHS research. Mentorship will be provided by training faculty, who are all members of the NIEHS Center for Research on Environmental Disease. The Center provides the cohesive and focused infrastructure that supports the EHS-SURP and provides a strong basis in EHS research. Under the direction of members of the training faculty, SURP students will conduct bench research on a defined, small project, related to the ongoing investigations in their mentor's laboratory. Students will also participate in didactic and career development activities designed to promote and prepare students for entry into graduate and professional education programs leading to EHS careers. The SURP has been continuously supported by a NIEHS T35 training grant entitled: Short Term Training for Minority Students, since 1996. Programmatic evaluations demonstrate that the SURP has been an important contributor to the career development of the participating students and has influenced their decision and ability to pursue graduate training leading to careers in scientific research and/or medicine.
This revised application for an Advanced Research Cooperation in Environmental Health (ARCH)
grant links the University of Texas at El Paso (UTEP), a Minority Serving Institution (MSI), with the
University of New Mexico Health Sciences Center (UNM HSC), a Research Intensive University
(RIU). The central research hypothesis is that children breathing air in the most polluted parts
of El Paso, TX, have an increased prevalence of asthma, which may be under detected in the
area's medically underserved Hispanic children. The project builds on the research strengths
and previous work of UTEP investigators in documenting significant air and soil pollution problems in El Paso, TX, and contiguous Juarez, Mexico, and on UTEP's recent successful programs in public health. The Core Research Project ties high-density (both spatial and temporal) air and soil quality data to the prevalence and intensity of asthma and respiratory distress in a cohort of 1200 households randomly selected from 100 stratified blocks in the El Paso community. The application has been completely revised with an improved environmental sampling design, development of a more expansive cohort, new environmental epidemiology expertise, and more productive interactions of all Pilot Projects with the Core Research Project. The Pilot Projects will monitor levels of pollutants, including PM2.5, carbon nanoparticles, toxic metals, polycyclic aromatic hydrocarbons (PAHs), atmospheric ozone, nitrogen dioxide (NO2), and volatile organic compounds (VOCs), and feed these data to the Research Core. In revised Project 1, lung function in children measured by Impulse Oscillometry (IOS) methods in a clinical setting will be compared to lung function measured with spirometry methods promotoras in the local communities. Project 2 is new and examines the relationship between indoor and outdoor air sample particle concentration and composition in a subset of the blocks in the Research Core Project. Project 3 is also new and will test for gases (NO2, ozone, and VOCs), again in neighborhoods tied to the Core. Revised Project 4 examines the hypothesis that the organic fraction of PM2.5 contributes to asthma via oxidized PAHs that activate cell signaling pathways important in inflammation and immediate type hypersensitivity leading to asthma exacerbations. Project 5 performs innovative studies on carbon nanotubes from local environmental sources. The ARCH Program is overseen by an Administrative Core and is supported by a Facility Core at UTEP.
DESCRIPTION (Taken from the Applicant's Abstract)
Baylor College of Medicine (BCM), the North Forest Independent School District
(NFISD), and the Harris County Department of Education (HCDE) propose to use
environmental health science as an integrated context for learning in six
highly disadvantaged, inner city elementary schools. The proposed project,
The Environment as a Context for Opportunities in Schools (ECOS), will use the
NIEHS-funded, interdisciplinary My Health My World educational program (which
was developed at BCM) as the backbone of a comprehensive curriculum that
integrates science, health, reading/language arts, mathematics, and social
studies.
Initially, ECOS will impact more than 3,900 students and 180 teachers in six
NFISD elementary schools (grades K-4). However, the aim is to produce and
evaluate a scalable and replicable model for integrating environmental health
sciences content across the elementary curriculum that can be applied in many
different settings. Project activities will involve partnerships among
researchers, educators, parents, community members, and administrators. The
specific aims are to (1) collaboratively design, implement, and evaluate an
instructional program for all six North Forest elementary schools that
integrates science, health, reading/language arts, mathematics, and social
studies around environmental health science themes, while promoting academic
achievement through development of student content knowledge and skills,
especially related to problem-solving and critical thinking; (2) improve
teacher practice through intensive summer and year-round professional
development on content and teaching strategies related to the proposed
instructional program and encouragement of team work, reflection,
collegiality, and self-assessment; (3) support school-wide reform of teaching
and learning through workshops and collaborative activities (such as science
festivals) involving teachers, parents, administrators, and community members;
(4) implement rigorous, hypothesis-driven evaluation strategies that examine
student content knowledge and problem-solving skills, student attitudes,
teacher development (particularly factors contributing to the development of
expert versus novice teachers), and whole-school reform; and (5) disseminate
the curricular/instructional model, evaluation instruments and methodologies,
and other research findings to schools in Houston, Texas, and throughout the
nation and to community stakeholders using multiple strategies.
DESCRIPTION (provided by applicant): Individuals exposed to the same environmental agent often respond differently due to additional factors such as genetics. Therefore, determining how human sequence variations (polymorphisms) influence the response to environmental exposures is key to understanding individual variability in disease susceptibility. The human p53 gene, which plays a critical role in the response to many cellular stresses, contains a common polymorphism that results in either an arginine (R) or proline (P) residue at position 72 of the p53 protein. Numerous epidemiological studies have associated this polymorphism with risk for developing various cancers and other diseases. However, different genotypes are associated with a predisposition for developing different cancers and in some cases there is conflicting data. Some laboratory studies suggest that the two p53 variants differ in their abilities to activate certain target genes while other studies suggest that the variants differ in their ability to induce apoptosis through a transcription-independent mechanism. Moreover, this polymorphism may affect the gain-of-function ability of mutant p53. A major problem with the interpretation of these functional studies is that all were done using artificial in vitro conditions and thus there is a critical need to model the R72P polymorphism in a way that is more physiologically relevant. This application is based on the hypothesis that the human p53 R72P polymorphism can be modeled in the mouse and that these models can be important tools for basic and translational research. Mouse models have been developed to study the role of specific mutations and sites of modification on p53, but no mouse model has been developed to study the R72P polymorphism. This may be because amino acid 72 is located in a region of human p53 that lacks homology to murine p53. To overcome this problem, we have used two different approaches to develop "humanized" mouse models for the p53 R72P polymorphism. Experiments proposed in this application will validate these mouse models with a focus on determining how the R72P polymorphism modulates apoptosis and skin cancer development. The long-term objective of this proposal is to use data from these mouse model studies to develop and test new hypothesis on the role of this p53 polymorphism in human health and disease. PUBLIC HEALTH RELEVANCE: Determining how small variations in the human genome (polymorphisms) influence the response to environmental exposures is key to understanding individual variability in disease susceptibility. Epidemiological studies suggest that a single nucleotide polymorphism (SNP) in the human p53 gene affects an individual's chances of developing various cancers but the underlying mechanism for this is not understood. This proposal seeks to use novel mouse models to study how this common p53 SNP modulates the development of human cancer and other environmentally related diseases.
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