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Record Count: 58
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DESCRIPTION (provided by applicant): Early- and late-phase estrogenic responses in the uterus have been recognized for more than 60 years, yet mechanisms involved in their regulation remain controversial. One concept is that an early events(s), occurring within the first 6 h, prepares the uterus for later (18-30 h) increase in DNA synthesis, cell proliferation and protein synthesis. An alternate view is that the late growth phase is a result of the continuous presence of a stimulus. Discussion of either concept usually makes the assumption that all of the responses are dependent upon ligand interaction with one of the two estrogen receptor isoforms (ER1 and ER2). However, increased gene expression following injection of estrogen or xenoestrogen, in mice lacking ER1, or in which all ER-activity has been suppressed by an estrogen-antagonist, ICI 182,780 (ICI), has shown this to be an oversimplification. In this regard, accumulating evidence suggests that estrogen regulates diverse but interdependent signaling pathways in uterine biology via ER- dependent and -independent manners. While our long standing hypothesis is that estrogenic certain early responses are ER-independent, late responses are ER1- dependent, and a cross-talk between the two phases is necessary for a full complement of estrogenic responses in the uterus. Our previous and preliminary observations suggest that among several such early genes, Bip and Sik-SP are induced by estradiol- 172 (E2) and kepone in the mouse uterus via ER-independent manner as a phase-I response. Moreover, we recently showed that Bip is critically necessary in mediating E2- or kepone-dependent estrogen signaling that involves ER1 functions. Our preliminary studies also indicated that E2 induces GPR30 and ERK1/2 phoshorylation without involving ER1. Furthermore, we observed that the administration of a selective inhibitor of ERK1/2 activation (SL327), 30 min prior to E2 injection abrogates early ER- independent genes, as well as uterine wet weights during the early and late responsive phases. In contrast, injection of the inhibitor 6 h after E2 injection did not alter late effects. Collectively, these results positioned us to study early and late estrogenic responses to determine molecular relationship between the phases. Our specific aims are to elucidate: 1. Early molecular signaling by E2 or kepone involving Sik-SP, Bip and GPR30 in the uterus. 2. Functional significance of Sik-SP and GPR30 in E2- or kepone- mediated late estrogenic responses in the uterus. Studies should yield new molecular insights involving Bip, Sik-SP and GPR30 during ER1-mediated estrogen signaling. Overall, the results will help us to define relationship and a molecular cross-talk between the two-phase estrogenic responses in the uterus. PUBLIC HEALTH RELEVANCE: Our specific aims are to elucidate: 1. Early molecular signaling by E2 or kepone involving Sik-SP, Bip and GPR30 in the uterus. 2. Functional significance of Sik-SP and GPR30 in E2 or kepone mediated late estrogenic responses in the uterus. Studies should yield new molecular insights involving Bip, Sik-SP and GPR30 regulation of ER1-mediated estrogen signaling. Overall, the results will help us to define the relationship and a molecular cross-talk between the two-phase estrogenic responses in the uterus.
DESCRIPTION (provided by applicant): When DNA is damaged, cells initiate a signaling cascade that results in cell cycle checkpoint arrest and repair of the damage or elimination of the damaged cells. One pathway that is activated following a double strand DNA break involves ATM and other DNA damage response proteins including CHEK2, Cdc25A, Plk3, BRCA1 and BRCA2. Several genes in this pathway, which contribute to genomic stability, have variant alleles that predispose to disease. This study focuses on mouse models and human cohorts who have had prior radiation exposure. The hypothesis is that variant alleles within the DNA damage response pathway may be benign or produce a modest risk, but that their adverse effects manifest more profoundly after exposure. One mouse model mimicks a CHEK2 variant that predisposes to breast cancer. Mice homozygous and heterozygous for this allele will be assessed for their inherent DNA repair capacity and whether they are more susceptible to disease and to mutagenesis, particularly to mitotic recombination and LOH before and after exposure. Other mouse models with deficiencies or variants in Plk3, BRCA2, and Cdc25A, have been made or are under construction. Additional models, dictated by the outcome of the human studies, will be produced. The human studies will utilize two unique cohorts with prior environmental exposure in the form of radiation who subsequently developed breast cancer. A control population consists of individuals who have experienced similar exposure but have not developed disease. The first cohort consists of patients who as children were treated with radiation for Hodgkin disease and years later developed breast cancer. The second cohort involves patients who worked at or lived in close proximity to the Fernald Uranium processing plant and have developed breast cancer. Specifically, we will ask whether variant alleles within the DNA damage response pathway, singly or in combination, are overrepresented in the affected population. When such alleles are identified, they will be modeled and tested in the mouse. Lastly, cells from affected individuals will be tested for genomic instability. In brief, this project assesses allelic variants within a pathway in two unique exposed populations and models the genotypes in mice. Since this pathway responds to damage induced by oxidative stress and since these alleles may sensitize to oxidative stress, the capacity of antioxidants to prevent disease in mouse models carrying variant alleles will be tested. PUBLIC HEALTH RELEVANCE: This project seeks to define genetic changes in a signaling pathway that contribute to susceptibility to breast cancer following environmental exposure. The program utilizes genetically modified mice that harbor variant genes that mimic gene variants found in human populations and that contribute to cancer. We will also ask whether such variants are over-represented in women who have had defined radiation exposure and now have developed breast cancer.
DESCRIPTION (provided by applicant): The long-term goal of this study is to understand the role that the mitogen-activated protein kinase (MAPK) signaling pathways play in the toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Dioxin is a widely spread environmental contaminant that exerts diverse species-specific toxic effects in animals and humans, including immune, reproductive and developmental toxicity, cancer, wasting syndrome and death. Dioxin toxicity is mediated by the activation of a cytosolic aromatic hydrocarbon receptor (AHR) that functions as a ligand-activated transcription factor and whose homozygous ablation protects mice from dioxin toxicity. Dioxin is non-genotoxic and, like other tumor promoters, is believed to exert its effects by promoting signaling pathways ultimately responsible for cell proliferation and cell survival. The (MAPKs) are the primary effectors of many of those signal transduction pathways. However, the molecular connections between dioxin-activated signaling pathways and AHR function have yet to be established. Dioxin-induced MAPKs play an important role in Ah receptor activation, because their suppression causes impaired AHR function. In addition, specific MAP kinase modules regulate Ah receptor function in a tissue specific manner. This proposal will test the hypothesis that dioxin-induced MAP kinase pathways regulate the activity and function of Ah receptor as a transcription factor. We will focus on the molecular identification of the signaling factors involved in dioxin action on MAPKs, the characterization of the mechanism of MAP kinase-mediated Ah receptor activation and the analysis of dioxin-induced AHR functions regulated by MAPKs in culture cells and in mice deficient in signaling factors of the MAPK pathways. To achieve these aims, we will use novel approaches that bring together an understanding of signal transduction pathways with the analysis of the molecular biology of the toxic response. Results from this work will further our understanding of cross-talks between dioxin-elicited biological pathways, will identify molecular factors critical for the diverse toxic effects of dioxin and will help characterize primary candidate targets for its prevention. Understanding the signaling mechanisms responsible for AHR activation by dioxin will provide a wealth of information immediately applicable to the study of the toxicity of the more than 400 environmental toxicants and Ah receptor agonists of which dioxin is the prototype.
Crisp Terms/Key Words: environmental exposure, cell proliferation, aromatic hydrocarbon receptor, mitogen activated protein kinase, gel mobility shift assay, SDS polyacrylamide gel electrophoresis, tissue /cell culture, autoradiography, protein structure function, phosphorylation, dioxin, ligand, western blotting, transcription factor, gene expression, biological signal transduction, genetically modified animal, laboratory mouse
DESCRIPTION (provided by applicant): Environmental exposures to polychlorinated biphenyls (PCBs) are known in humans as well as lab animals to cause immunosuppression, thyroid disease, endocrine disruption, and damage to the central nervous system. Not all humans or laboratory animals respond similarly to the same dose -indicating interindividual genetic differences. In rodents to elicit these pathologies, planar PCBs must bind to, and activate, the aryl hydrocarbon receptor (AHR). Despite this overriding role for planar-PCB-mediated AHR activation in toxicity, the AHR up-regulates CYP1A2, which in liver sequesters and protects distant tissues against planar PCBs. Both the AHR and CYP1A2 are polymorphic in humans: the AHR exhibits >12-fold differences in ligand-binding affinity; liver basal CYP1A2 shows >60-fold differences in subjects having no known exposure to inducers. With regard to fetal exposure, our studies in mice demonstrate that risk of birth defects by planar TCDD depends on the high-affinity AHR and is also greatly increased in fetuses carried by dams that lack CYP1A2. PCBs represent mixtures having many dozens of different congeners; which congener is toxic, and the rates of uptake, metabolism and excretion are difficult to determine in humans, and most studies in lab animals look at a single congener. We have studied mice with the high- (Ahrb, B6) vs low- (B6.D2-Ahrd) affinity AHR, and with or without the Cyp1a2 gene. Using these mice, we hypothesize that Ahrb fetuses carried by Cyp1a2(-/-) dams will be most susceptible, and Ahrd fetuses carried by Cyp1a2(+/+) dams most resistant, to deficits in learning, memory, and other behaviors caused by planar PCBs. For the funding period, we propose to: [1] determine tissue distribution of each of eight PCB congeners (most relevant to humans) given as a mixture -comparing B6 vs B6.D2-Ahrd, and Cyp1a2(+/+) vs Cyp1a2(-/-) dams and their offspring; and [2] evaluate the in utero and lactational effects of this orally administered PCB mixture on learning, memory, and other behaviors in offspring of these treated dams, starting at postnatal day 60. These studies will define the impact of a fetal basis for adult disease. The Ahr and Cyp1a2 genotypes in these mice represent the extremes for variability of these two genes in the human population. There exist genetic differences in mouse (and human) populations, which represent a gradient of at-risk individuals. Project Narrative: Polychlorinated biphenyls (PCBs) are widespread persistent organic pollutants linked to numerous human health problems, including learning and memory deficits in children of exposed mothers. The Ahr and Cyp1a2 genotypes in our mouse models represent the extremes for variability in these two genes in the human population, and both genes likely play a role in susceptibility following PCB exposure. These studies will define the fetal basis for adult disease and help to identify individuals at greatest risk of PCB-induced neurotoxicity.
DESCRIPTION (provided by applicant): The long-term objective of this proposal is to understand the molecular mechanisms underlying the biological responses to dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin; TCDD) exposure. TCDD, the prototypic dioxin and a model for many other organochlorinated compounds, produces many apparently unrelated biological effects, ranging from chloracne in humans to developmental teratogenesis, tumor promotion, thymic atrophy, wasting syndrome and death in laboratory animals. In addition, TCDD, a rodent carcinogen, is strongly suspected of being carcinogenic also in humans. The molecular basis of the biological effects of TCDD is largely unknown. Dioxin is a ligand for the aromatic hydrocarbon (Ah) receptor (AHR), which, as a dimer with the Ah receptor nuclear translocator protein ARNT, mediates the transcriptional activation of genes in the CYP 1 family of cytochrome P450 monooxygenases. However, activation of the CYP1A1, CYP1A2 and CYP1 B1 genes, although one of the best characterized effects of Ah receptor activation by TCDD, does not adequately explain the diversity of TCDD effects. Our recent global expression profiling analyses of human hepatoma cells shows that exposure to dioxin induces or represses a total of more than 300 genes, with repression being the more frequent. Induction may readily be explained by the transactivating potential of the AHR, but gene repression is a novel effect of the activated AHR that is uncharacterized at the molecular level. The goal of the experiments proposed here is to define and characterize the regulatory interactions between the activated AHR and other transcription factors, co-regulators and chromatin remodeling factors responsible for the effects of dioxin on gene expression. The major objectives of this work are, (1) to define the role of discrete domains of the AHR in gene regulation; (2) to use proteomic analyses to identify AHR coregulatory partners in gene induction and repression; and (3) to clone the promoters of AHR regulated genes and characterize their response to dioxin exposure. Results from these experiments will be crucial for our understanding of the long-range biological consequences of exposure to dioxin and to other organochlorinated compounds and will help formulate an adequate rationale to deal with health problems arising from an ever-increasing exposure to these environmental agents.
DESCRIPTION (provided by applicant): The long-term goal of this project is to identify and characterize the gene(s) responsible for interindividual differences in response to cadmium (Cd). Environmental Cd is absorbed by the small intestine and lung, is bound to metallothionein and glutathione in hepatocytes, and is deposited in the kidney. The kidney is the major target organ for human and lab animal Cd exposure. During the current funding period, we identified the Slc39a8 gene as the Cdm locus responsible for Cd-induced testicular necrosis. Slc39a8 encodes the ZIPS transporter protein, which we show is a high-affinity apically-oriented rogue Cd/HCO3- cotransporter. ZIPS expression in culture increases Cd influx and sensitizes (>30-fold) the cells to Cd. ZIPS is expressed in a cell-type-specific manner and is highest in alveolar and tubular epithelial cells in the lung and kidney, respect- tively. A BAC-transgenic (BTZIP8) mouse line containing 2 + 1 additional copies of the Slc39a8 gene shows a gene-dose-dependent increase in ZIPS mRNA in all tissues tested, and is acutely sensitive to Cd-induced kidney dysfunction. Slc39a8 is one of 14 members of a solute-carrier (SLC) family of metal transporters; Slc39a14 shares the most recent ancestry with Slc39a8; ZIP14 exists as two peptides, ZIP14A and ZIP14B, as a consequence of alternate exon splicing. The ZIP14s are expressed highest in small intestine and liver and have transport and toxicologic properties similar to ZIPS. Our hypothesis is that the ZIPS and ZIP14 transporters are the principal mediators of Cd-induced renal dysfunction. For the next funding period, we propose to: [1] characterize the transport and physiological properties of ZIPS, ZIP14A and ZIP14B in vitro; [2] study the physiological role of ZIPS in Cd-induced renal dysfunction using our BTZIP8 overexpresser and Slc39a8(-/-) knockout mouse models; and [3] develop and characterize similar mouse models for Slc39a14 These studies will improve our understanding of heavy-metal toxicity and may lead to uncovering new targets that might be useful for preventive strategies as well as therapeutic intervention in heavy-metal diseases.
DESCRIPTION (provided by applicant):
The maintenance of genomic stability in eukaryotes is relentlessly challenged by DNA damage. In order to understand the basis of damage tolerance and avoidance of deleterious effects, it is important to grasp not only the nature of such DNA damage and its processing pathways in cells, but also to have a clear understanding of how cells manage the lesion repair in the highly compact and tightly regulated chromatin milieu of the eukaryotic nucleus. This proposal is based on the premise that the specific biochemical pathways, relying on wide-ranging inter-molecular cross-talk between the pathway components, operate at a higher efficiency under conditions of macromolecular crowding and sequentially recruit specific factors for access a s well as repair of genotoxic lesions within mammalian genome. The proposal builds on our recent observations that support a strong link between DNA repair and p53 mediated and other related regulatory pathways. The overall experiments are designed to address the specific hypothesis that cellular DNA damage recognition and transcription factors, congregate at DNA lesions sites, open the chromatin via histone acetylation and initiate the key pre-incision events of nucleotide excision repair (NER). The proposed work will utilize the PI's established expertise in relevant, cellular, molecular, biochemical, and immunological technologies to undertake the following specific aims. (1) To assess the nature and extent of contribution of damaged DNA binding protein, DDB, in NER through quantitative assessment of NER sub-pathways in human cells of defined protein status. (2) To demonstrate the role of DDB for sequential recruitment of initial recognition and downstream core NER factors through spatial and temporal co-localization in situ. (3) To determine the participation of histone acetyl transferases in damage recognition and excision via qualitative protein cataloging and targeted recruitment and physical association of interacting factors. (4) To establish the histones acetylation as a direct and key consequence of DNA damage by placement of acetylation events exclusively at the lesion sites in vivo and in vitro. These studies will provide important insights regarding the dynamics of DNA damage recognition and processing through identification of cross-talking events and critical factors that influence the maintenance of genomic stability so important to overcome the hazardous health effects of genotoxic environmental exposures.
DESCRIPTION (provided by applicant): Prompt and error-free repair of critical genomic alterations of toxicant exposures is vital to normal survival of living organisms. During the previous project period, we have shown that global genomic repair lends itself to elaborate regulatory control for orchestrating the "comings and goings" of multiple repair factors. This continuation project will extend the scope of these studies along a similar overall theme. The proposal is based on the premise that in response to genotoxin exposures, several seemingly independent cellular pathways converge to function in tandem for the distinctive recognition and effective excision of DNA lesions. The specific hypotheses addressed are: (i) entire repair edifice is composed from the initial regulated attaching of DNA damage binding protein that impinges on recruiting subsequent core repair factors, (ii) cellular ubiquitin/proteasome apparatus intimately participates in sequential clearance of repair factors that are tightly bound to damage sites, and (iii) the involved repair protein complexes allow bridging between diverse pathways through multi-acting pleiotropic factors. The specific aims will focus on: (1) establishing the biochemical/molecular basis for selective damage recognition and recruitment of DDB containing E3 Ub-ligase complex to diverse DNA lesions, (2) defining the role of Ub-mediated proteolysis in lesion hand-over during repair processing, (3) delineating the nature and function of DNA damage dependent XPC modifications, (4) establishing the molecular basis for differential role of hHR23A/B in regulation of XPC ubiquitination/degradation and UV-induced p53 response, and (5) understanding the role of 19S proteasomal components and other cellular deubiquitinating enzymes in regulation of XPC stability. The experimental studies will continue to concentrate on genomic modifications induced by a representative physical (UV radiation) and a chemical (anti-BPDE) genotoxic carcinogens and human cell lines representing multiple organ systems, e.g., skin fibroblast, liver hepatocytes, breast and lung epithelium. State-of-the-art biochemical, molecular and cellular methodologies, mostly established in the PI's laboratory, will be applied along with the newer evolving technologies to accomplish these specific objectives. The studies will provide seminal insights into the molecular responses of xenobiotic action and processing of resultant genotoxic damage which has crucial implication in risk assessment of human environmental exposures.
Zeranol (Z) is a non-steroidal agent with estrogenic activity (E2) that is used as an anobolic growth promoter
in the U.S. beef industries. Consumption of bio-active Z containing-beef has a potential risk in human normal
and cancerous breast cells. Previous work suggests that PTP gamma (PTPG) is an E2-regulated breast
cancer suppressor gene. Z is capable in down-regulating the PTPG both in mRNA and protein levels in
human normal and cancerous breast tissues and cells. The overall HYPOTHESIS to be evaluated is that the
consumption of beef products, derived from Z-implanted beef cattle may possess a potential health impact
on human consumers, particularly with respect to reproductive endocrinology and hormone-sensitive breast
tissues.
Specific Aims: (1): Define epigenetic and gene expression alterations in primary cultured normal human
breast epithelial cells, stromal cells, and tissues exposed to serum and meat extracts collected from Zimplanted
beef heifers. (2): Define DNA methylation changes in the estrogen inducible mammary tumor ACI
rat model exposed to bio-active Z-containing diets formulated from Z-implanted beef heifers. (3) Investigate
mammary tumorigenesis in the estrogen inducible mammary tumor ACI rat model after long-term
consumption of low level of bio-active Z containing experimental diets formulated from meat collected from
Z- implanted beef heifers.
The team of researchers assembles expertise in 3 areas to systematically study the mitogenic effects of Z in
reproductive and hormone sensitive breast. These include modeling of in vitro and in vivo effects of Z (Lin),
mapping and quantification of global and unique epigenetic alterations in gene promoters (Huang & Yan),
and comparative quantification of global gene expression and identification of biomarkers (Wick).
Significance: The proposed experiments will provide a clear linkage between bio-active dietary Z and PTPG
down-regulation to eventual tumorigenesis via epigenetic regulation of the expression of PTPG and other
tumor relevant genes. To translate our findings from bench top to bedside, selected biomarkers uncovered
from the in vitro experiments will be further validated by proteomic analysis. It is our intent to provide
comprehensive data (exposure levels during Z-implantation and after Z withdrawal from the beef heifers) to
raise awareness of environmental exposure to the estrogen mimic, beef growth promoter, Z.
DESCRIPTION (provided by applicant): The importance of somatic mutation in the genesis of cancer and other diseases is undisputed. However, the extent to which loss of heterozygosity (LOH), as a consequence of mitotic recombination, contributes to the frequency of spontaneous mutation has been under-appreciated. Using a mouse model that is heterozygous at Aprt we have previously reported that in vivo spontaneous mutation frequencies at Aprt can approach 10-4 and that up to 80 percent of these events can be due to mitotic recombination. The global mutation frequencies, in fact, are even higher given that all loci between the point of crossover and the reporter locus are also affected, and that mitotic recombination can occur between all autosome homologs. The goals of this program are to further exploit our Aprt heterozygous model and to develop additional Aprt-derived models to ask questions regarding local events at sites of double strand breaks, and to determine whether different pathways to mutation/repair are preferred in different isogenic cell types. An emphasis will be placed on embryonic stem (ES) cells and adult stem cells since they have requirements beyond those of somatic cells for preserving the integrity of their genomes. We have already reported that ES cells suppress mutation and mitotic recombination by as much as 100 fold compared with isogenic mouse embryo fibroblasts (MEFs), one mechanism to protect their genomes. We and others have also shown that ES cells are hypersensitive to DNA damage, and that they lack a G1 checkpoint, presumably facilitating death and removal of cells that have acquired a mutational burden. This represents a second mechanism by which ES cells protect their genomes. We have identified the signaling pathway that is compromised in ES cells after DNA damage, reconstituted it, and shown that reestablishment of a G1 arrest after challenge protects the cells from apoptosis. We wish to ask whether adult stem/progenitor cells behave more like somatic cells or ES cells with respect to cell cycle regulatory mechanisms and mechanisms to suppress mutation. The role of adult stem cells in the genesis of tumors is an important question that is not yet resolved. We will use our Aprt null mice and intestinal epithelium to ask whether mutations in the mouse gut arise exclusively in the crypt base, site of the progenitor cells or whether they also arise elsewhere within the crypt. This experiment should provide evidence for or against the "top down" model of colon cancer that has been proposed.
DESCRIPTION (provided by applicant): Polycyclic aromatic hydrocarbons (PAHs), nitrosamines, and N-heterocyclics are present in combustion products, e.g. grilled foods & cigarette smoke. Cytochromes P450 1A1 & 1B1 (CYP1A1, CYP1B1) are responsible for the metabolism of numerous PAHs, the prototype of which is benzo[a]pyrene (BaP). CYP1A2 is responsible for metabolizing nitrosamines and N-heterocyclics, but also PAHs (in particular, BaP) to a lesser extent. Using Cyp1a1(-/-) and Cyp1b1(-/-) knockout mice, we have shown that CYP1A1 is more important in detoxication than metabolic activation, whereas CYP1B1 causes metabolic activation of BaP to unwanted reactive intermediates. In other words, CYP1A1 is more good than bad in the intact mouse ingesting BaP, and CYP1B1 is more bad than good in the intact mouse administered PAHs by various routes. The importance of mesenteric lymphatics vs. the portal system (mesenteric blood vessels, liver, bile) is not known for oral BaP. This lab now has seven-all three single, all three double, and the one triple-Cyp1 knockout mouse lines. Our hypothesis is: lymph BaP uptake and CYP1B1 in distal tissues (e.g. immune cells, spleen, and bone marrow) are the principal causes of oral BaP toxicity, whereas inducible CYP1A1 in liver and intestine is the principal cause of BaP detoxication. In this proposed project, we therefore will: [a] identify and determine the amounts of metabolites vs. unchanged parent BaP in mesenteric lymph, portal vein blood, liver, and bile in wild-type and all seven Cyp1 knockout mouse lines, and the role and mechanism of chylomicrons in delivering BaP to target organs; [b] generate liver- and intestinal epithelium-specific Cyp1a1 conditional knockout lines; [c] replace the Cyp1b1 gene (in the genome) with the Cyp1a1 gene, and vice versa; and [d] repeat our BaP pharmacokinetics studies (see [a]) in these four newly generated mouse lines. Understanding the tissue- specific roles for each of the three CYP1 enzymes in the intact mouse receiving oral BaP will provide us with a greater understanding of BaP detoxification vs. metabolic activation. We expect this knowledge will provide a blueprint for understanding the mechanisms of elimination vs. dissemination of ingested BaP and will be informative in clinical studies in which we would determine which haplotypes of these three human genes might be associated with resistance vs. sensitivity to PAH-induced toxicity and cancer.
DESCRIPTION (provided by applicant): The contribution of residential hazards to children's health is poorly defined, but it is clear that some of the most prevalent disease and disabilities among U.S. children - including injuries and lead poisoning - would decline if such hazards were eliminated. Residential hazards also contribute to social disparities in children's health. This revised community-based participatory research application - a partnership of the Cincinnati Children's Hospital and the National tenter for Healthy Housing - would extend the follow-up of an ongoing 402-person birth cohort (n=358 in the randomized trial) designed to test the efficacy of interventions to control residential hazards in children, from 2 years of age through 5 years. This extension is critical to fully evaluate the efficacy of reducing lead and injury hazards on children's intellectual abilities, behavioral problems and injuries during the peak vulnerability of early childhood. The central hypothesis of this application is that housing interventions will reduce the prevalence of lead toxicity and residential injuries in a racially diverse sample of children. The aims and hypotheses of this application are: 1.0 Determine the efficacy of lead hazard controls on children's blood lead levels and their risk for learning and behavioral problems. 1.1 Levels of lead in dust, soil and water will be significantly lower for housing units in the lead treatment arm compared with the injury control arm at 36, 48 and 60 month home visits. 1.2 Children who are assigned to the lead treatment arm will have lifetime blood lead levels that are >20% lower than children assigned to the injury treatment arm at 5 years. 1.3 Children in the lead treatment arm will have higher IQ scores at 5 years than children in the injury treatment arm. 1.4 Children in the lead treatment arm will have fewer behavioral problems at 5 years, especially measures related to ADHD, than children in the injury treatment arm. 1.5 Higher lead exposure, measured during pregnancy and early childhood using maternal blood, cord blood, meconium and children's blood, is inversely associated with IQ scores and directly associated with behavioral problems at 5 years in children with postnatal maximal blood lead levels < 5 /jg/dL 2.0 Determine the efficacy of comprehensive housing modifications on children's risk for injuries. 2.1 Children who are assigned to a multifactorial, housing intervention to prevent injuries will have 30% fewer unscheduled medical visits for housing injuries compared with children in the lead treatment arm. This is the first trial to test the efficacy of a multifactorial intervention for the primary prevention of two of the most prevalent causes of disease and disability in US children - lead toxicity and residential injuries. If efficacious, policy could be developed to reduce disease and disability linked with these hazards. It would also help resolve the ongoing controversy about the adverse consequences of low-level lead toxicity at blood lead levels previously thought to be safe. Our partnership with the National Center for Healthy Housing will enhance our ability to translate and disseminate the research results to affected communities locally and nationally.
DESCRIPTION (provided by applicant): Among the fundamental principles of chemical mixture toxicity that remain to be clarified are the relationships between the type of combined effect obtained and each of the following: time-dependent toxicity, the molecular site or sites of action of the agents, and chemical reactivity mechanisms. The work proposed in this application is designed to help determine these relationships, thereby providing a sounder fundamental basis from which to perform risk assessments for chemicals in combination. The approach involves mixture toxicity testing of various soft electrophiles using a previously developed Microtox(r).-based mixture toxicity assay. This assay utilizes the bioluminescent marine bacterium Vibrio fischeri. The chemicals evaluated in combination, soft electrophiles, are agents that preferentially react with specific molecules within proteins. The toxicity of soft electrophiles, both singly and in combination, is of current concern due to their use as fragrance additives in common household products and various industrial processes that can lead to skin sensitization and environmental contamination. By testing chemicals in binary combinations: a) from within the same reaction mechanism, or b) across different reaction mechanisms, and c) across varying reactivity rates (from very fast to very slow and non-reactive), it will be possible to more completely develop/expand the theoretical framework for understanding chemical mixture toxicity from a mechanistic perspective. Preliminary data suggests that mixture toxicity often resembles the dose- addition combined effect, generally, but with results for many combinations that significantly deviate from dose-addition -the combined effect expected for agents working at the same, single molecular site of action. Upon completion, the project will have generated a wealth of toxicity data that will be used to define the relationships between the combined toxic effects obtained, the time-dependent toxicity of the individual agents, the molecular site(s) of toxic action of the agents and their chemical reactivity mechanisms. PUBLIC HEALTH RELEVEANCE: The relevance of this research to public health in that it will evaluate the toxicity, both alone and in mixtures, of specific chemicals that cause skin sensitization - an allergic reaction occurring in sensitive people due to physical contact (e.g., skin, mucous membranes) with these agents. Additionally, many of these chemicals are used in industrial processes and, therefore, have the potential to be harmful to people who produce, transport, or handle them. Environmental releases of these chemicals are possible, which could also affect public health.
Polyaromatic hydrocarbons (PAHs) are significant pollutants at Superfund sites. They are often toxic and may be carcinogenic. Consequently, research is needed on practical methods to remove the PAHs from contaminated groundwaters. PAHs are usually accompanied at these sites by heavy metals such as arsenic and cadmium. Unfortunately, little is known about the interactions between PAHs and heavy metals and the effect the presence of these metals may have on PAH bioremediation. Complex mixtures of toxic organics and metals are difficult to study and have, therefore, been largely ignored. The overall goal of this research s to gain an understanding of the transport and biodegradation mechanisms of complex mixtures of toxic
pollutants at Superfund sites. We will be studying a novel groundwater treatment systems known as a "biowall" or permeable bio-barrier system. Determining the role of biofilm in permeable bio-barrier systems is crucial to the design of more efficient treatment systems since the mass transport mechanisms, structural forms, and biodegradation processes in soil biofilms are not well understood. The engineering component will focus on design and evaluation of mulch-based biowalls and development of pollutant monitoring microelectrodes for application in studying the biodegradation of PAH-metal mixtures in water aquifers at Superfund sites. The potential use of fungi in the biowall will be examined. Research will be conducted in a
simulated Superfund testbed that replicates a contaminated groundwater system. The successful monitoring of biofilms and pore water at Superfund sites will also improve process efficiency in bioremediation treatment. Therefore, microelectrodes developed in previous SBRP research will be modified for field use and tested in the testbeds.. In addition, a new MEMS-based heavy metal analyzer for use in the field will be developed. The result of this research-will be a better understanding of how complex mixtures of toxic materials can be bioremediated at Superfund sites so as to reduce the potential impact of these PAHs and
heavy metals on human health.
Because of their intimate association with soil contaminants, we HYPOTHESIZE that the diversity
[measured as richness and evenness) of the metabolically-active fraction of the soil bacterial community
serves as a biomarker of the threat to human health from PAHs and heavy metals polluting Superfund sites.
To challenge this hypothesis, we will address three SPECIFIC AIMS: (1) Identify correlations between the
results of molecular biology-based measures of bacterial diversity and analytical measures of PAHs and
heavy metals during soil ageing and bioremediation of mock Superfund sites; (2) Demonstrate novel
molecular biology-based assays to assess the diversity of the metabolically active fraction of the bacterial
community in situ; and (3) Demonstrate ecological models to predict soil bacterial community diversity under
the influence of mixtures of heavy metals and PAHs. The APPROACH includes: (i) operating mock
Superfund sites; (ii) T-RFLP and (iii) whole cell FISH to measure bacterial diversity; (iv) identifying
predominant bacterial populations using clone libraries; (v) evaluating the impact of PAH and heavy metals
on ribosome genesis; (vi) identifying heavy-metal responsive genes using microarrays; (vii) evaluating
metagenomics of PAH degradation; (viii) evaluating real-time PCR to quantify heavy-metal and PAHresponsive
genes; (ix) adapting ecological models to predict bacterial diversity; (x) comparing experimental
measures of diversity with model predictions; and (xi) evaluating optimum nutrient amendments predicted
with the resource-ratio theory. This project is INNOVATIVE because we are using molecular biology-based
measures of bacterial diversity as a diagnostic tool to predict the threat to human health at Superfund sites
(i.e., bacteria as sentinels for pollution). We expect that the positive findings of this study can be used to
evaluate the potential for success of bioremediation strategies as well as to establish an effective endpoint of
bioremediation (i.e., when appropriate bacterial diversity has been re-established and the threat to human
health significantly reduced).
DESCRIPTION (provided by the applicant):
Hexavalent chromium[Cr(VI)]exists at the Superfund sites where it poses major health concerns to humans. Bacteria-mediated in situ transformation of water soluble and toxic Cr(VI) to less soluble/toxic Cr(III) represents a promising method to contain and treat Cr(VI). However, the molecular mechanisms by which bacteria reduce Cr(VI) remain undetermined. Reduction of Cr(VI) by dissimilatory iron (Fe) reducing bacterium Shewanella oneidensis results in formation of nano-sized Cr(III) particles on the cell surfaces. We hypothesize that cell surface-exposed cytochromes MtrC and OmcA of S. oneidensis are involved in the formation of Cr(III) by reducing Cr(VI) directly as Cr(VI) terminal reductases and indirectly as Fe(III) oxide terminal reductases to produce Fe(II) that in turn reduces Cr(VI). We plan to use nanoscale AFM correlated SERS imaging microscopy (AFM-CSIM) developed by us along with other methods to test our hypothesis. AFM-CSIM can simultaneously determine the locations of cytochromes and nano-sized metal particles as well as their oxidation states on the bacterial surfaces at nanometer-resolution. Co-localization of MtrC and OmcA with nano-sized Cr(III) and Fe(III) particles by AFM-CSIM will provide direct evidences to support our hypothesis. Other approaches (e.g. immuno-gold localization with scanning electron microscopy and the measurements of Cr(VI) reductase activity of purified MtrC and OmcA) will be used to validate the AFM-CSIM results. Successful completion of this project will help not only achieve our long-term goal of understanding the molecular mechanisms by which bacteria reduce Cr(VI), but also develop science-based solutions to mitigate the toxic effects of Cr(VI) at the Superfund sites.
DESCRIPTION (provided by applicant): Microbial biodegradation is an effective way to protect human health from polluted environments contaminated with highly toxic and carcinogenic chemicals such as polycyclic aromatic hydrocarbons (PAHs). Persistence of PAHs at these sites is due to the resistance of high m.w. PAHs (4-5 ring) to biodegradation by natural microflora. This necessitates the use of appropriate bioremediation agents and strategies that can enhance biodegradation of these recalcitrant PAHs. Our focus is on the wood-rotting white rot fungus Phanerochaete chrysosporium. Despite its extraordinary biodegradation potential, little is known on the enzymatic processes and conditions required for effective field application of this fungal agent in bioremediation of resistant PAHs in the environment. Using functional genomic approach, we have identified a subset of PAH-inducible P450 monooxygenase genes in P. chrysosporium, which are expressible under more varied nutrient conditions unlike the previously characterized peroxidases. We will test our hypothesis that an effective bioremediation activity of the white rot fungus toward higher PAHs (4-5 ring) can be achieved in a biphasic approach involving optimized initial expression of its promising P450 monooxygenases (for initial oxidation) preceding the expression of ligninolytic enzymes (for subsequent biotransformation/ mineralization to CO2) with nutrient depletion such as on lignocellulose. The overall goal of this research is to gain an understanding of the promising P450 system for use in P450-initiated biodegradation of PAHs in this organism as a basis for developing effective bioremediation strategies. The specific aims are (1) Functional expression of the candidate P450s to select the promising PAH-oxidizing P450(s) and generate P450 fungal biocatalyst(s); (2) Investigate catalytic activity of the developed P450 biocatalyst(s) and further biodegradability and toxicity reduction of the P450-oxidation products of PAHs by white rot fungus. (3) Optimize conditions for enhanced expression of the newly-identified PAH-oxidizing P450 vis-a-vis the ligninolytic peroxidases using varied treatment conditions; (4). Evaluate the optimized conditions and developed P450 biocatalysts for bioremediation of PAHs by the white rot fungus. The studies are expected to provide a knowledgebase for development of effective bioremediation technologies for recalcitrant PAHs based on the use of white rot fungus. PUBLIC HEALTH RELEVANCE: Microbial biodegradation is an effective way to protect human health from polluted environments contaminated with highly toxic and carcinogenic polycyclic aromatic hydrocarbons (PAHs). This proposal will develop appropriate fungal biocatalysts and strategies for PAH bioremediation.
Breast cancer is the second leading cause of cancer deaths among women in the U.S. today, claiming 40,000 lives per year. Several factors, including societal influences on childbearing, diet, and environmental chemicals are believed to be impacting the high rate of breast cancer in this country. Reproductive factors are particularly important in the increased incidence in the U.S., two major factors being the depression in the age of menarche and the extension in the time before first full-term pregnancy. Reasons for the depression in the age of menarche are not fully understood, but appear to be due in part to diet, whereas the delay of childbearing is primarily social. Because the contemporary development and maturation of the breast is
during this extended time from menarche to pregnancy, there is further time for toxic influences to have an opportunity to induce tumor formation. Our hypothesis is that diet, in particular fatty acid and phytoestrogen composition and quantity, during the perinatal period and childhood determine the level of adiposity and regulate the hormonal milieu in young children. Through leptin and insulin-like growth factor, adiposity determines the pathway of puberty as being driven primarily by the adrenal gland (adrenarche) or ovaries (thelarche), the latter being associated with early menarche and subsequent risk of breast cancer. In prospective cohorts from in utero to age 3 and age 6 to 12, we shall test the association of diet and adiposity, as well as environmental agents, eg endocrine disruptors and carcinogens, and qualities of the psychosocial environment, with pubertal pathway. Through analysis of hormones, growth factors, and aromatase we shall examine mechanisms through which diet may mediate these effects. In rodent studies we shall test the hypothesis that dietary fatty acid and phytoestrogen composition in utero and early life alter puberty and mammary gland maturation. We further predict that these factors alter the periods of life at which mammary glands are most susceptible to carcinogenic insults. Using the power of gene expression arrays we shall define characteristics of initiated mammary epithelial cells that can be used to examine endogenous and exogenous compounds for their carcinogenic potential and better define initiated mammary cells in animal models and human studies.
DESCRIPTION (provided by applicant)
This application requests a 5-year renewal of the Center for Environmental Genetics (CEG). The unifying research focus of the Center is to study the impact of genetic diversity on individual susceptibility to deleterious environmental agents. The CEG has a long 15-year history of accomplishments in environmental genetics that helps shape one of the nation's current top priorities in environmental health sciences (EHS) research. The investigators will expand their pioneering efforts and propose the following aims for the next funding period. Aim 1: To promote research in gene-environment interactions in human health and disease by emphasizing the use of environmental exposures in relationship with genetic variations and epigenetic reprogramming to inform pathophysiologic endpoints. Aim 2: To hasten translation of basic biomedical EHS research and population-based studies into clinical practices for disease prevention, diagnosis, and prognosis. Reciprocally, Center activities will afford opportunities for novel hypotheses to be generated from human based studies to be tested in laboratory settings. Aim 3: To foster the development of sensitive pathophysiological markers for the assessment of internal dose and improve measures of body burden; to undertake the discovery predictive makers so that the effects of early life exposure on later-life disease risk could be anticipated and minimized through innovative intervention measures. Validation of these markers through quantitative and qualitative measurements of phenotype or clinical outcomes will be an integral part of this aim. Aim 4: To attract and train the next generation of biomedical EHS investigators and clinicians with a strong desire to apply EHS in their clinical practices through partnership of the investigators' comprehensive Career Development Program (CDP) and a newly established Master Degree in Clinical Training program. A Pilot Project Program will function to continue support young investigators till they attain independence in EHS research, and to recruit new-to-EHS scientists. Three Facility/Service Cores will facilitate attainment of the goals: 1) the Integrative Technology Services Core will inject leading edge technologies into EHS research; 2) the Integrative Health Services Core will help manage and share epidemiological and clinical databases, support tissue procurement, and development of clinical protocols; and 3) the Informatics Core will support all levels of data-mining and hypothesis-creation opportunities from various systems biology and clinical investigations. The overarching objective of the Center is to improve human health through effective integration of basic, clinical and epidemiological research, germane to gene-environment interaction, toward the ultimate goal of delivering "personalized" medicine and devising public health initiatives for susceptible populations. The multiplicity of expertise and strong fund holding amongst membership, and the unyielding commitment from institution leadership constitute the elements of success in this application.
BACKGROUND
The University of Cincinnati, College of Medicine (UCMC) was established in 1819. The Kettering Laboratory of Applied Physiology at UCMC was established in 1930. In 1965, this Laboratory became the Department of Environmental Health (DEH). The Center for Environmental Genetics (CEG) is housed in the Department of Environmental Health at the University of Cincinnati, College of Medicine. This is the third competing continuation application for the University of Cincinnati Center for Environmental Genetics, funded initially in April 1992. The first Center Director was Dr. Daniel Nebert (1992-1997), the second was Dr. Marshall Anderson (1997-2005), the third (interim) was Dr. Alvaro Puga (2005-2006), and the fourth and current Director is Dr. Shuk-mei Ho (2006-present). There is a close collaboration between University of Cincinnati, College of Medicine and the Cincinnati Children's Medical Center (CCHMC); most members of the Center for Environmental Genetics have affiliations in these two Medical Centers.
This application is a revised competing continuation application which includes changes suggested from the previous review. The theme of the Center for Environmental Genetics is to develop an understanding of the complex relationships between genetic susceptibility factors and environmental exposures, as well as alterations in genetic-epigenetic interplay under environmental influences, and to apply the understanding of these concepts to the study and alleviation of the pathogenesis of environmentally-induced diseases. The focus of the Center is to understand how exposure to environmental factors interacts with genes, genetic variants, and epigenetic modifiers, either to preserve health or to cause disease.
ESSENTIAL CHARACTERISTICS
Strategic Vision and Impact on Environmental Health
DESCRIPTION (provided by applicant): Organochlorine compounds (OCs) are toxic, carcinogenic, and ubiquitous in the environment. Oral ingestion is the principal route of entry into the human body for many OCs. Because of the lipophilicity of OCs and many of their metabolites, they are preferentially stored in adipose tissue, but are also found in liver, brain, and other organs and tissues. Although oral ingestion is the initial step in the accumulation of OCs in animals and humans, the processes involved in absorption from the intestine and early transport in blood and lymph are not well characterized. We propose studies that will shed light on these important steps in absorption and transport to provide a basic framework that can lead to understanding the ways that nutrients influence the absorption processes. In Specific Aim 1, we will determine the mode of absorption and association of OCs with carriers in lymph and the portal blood and whether this is affected by the amount and the type of lipid fed. We demonstrated that labeled hexachlorobenzene (HCB) carried in chylomicrons is cleared from the circulation faster than the labeled fatty acid (FA) as part of the triglyceride (TG). To explain this finding, the labeled HCB must not have been dissolved in the TG lipid core of the chylomicron. If it is, then its rate of removal from the circulation should be slower or at best equal to that of the labeled TG. Consequently, in Specific Aim 2, we will determine the mode of delivery of labeled OCs to the tissues (especially the adipose tissue) by proteins, phospholipids, and chylomicrons (CMs). We will also determine the partitioning of HCBs between chylomicrons and any membranes it comes in contact with e.g., red blood cell membrane. Lastly, in Specific Aim 3, we will determine if the relative importance of vehicle (albumin, lipid, or lipoprotein) carrying the labeled OC in blood is altered as a result of weight loss or weight gain (i.e. changes in body fat). The proposed research is important because of the biological and clinical importance of OCs in health and disease and they provide considerable new insights into how OCs are carried and delivered to the various organs in the body.
DESCRIPTION (provided by applicant): The increasing rates of breast cancer occurring in developed countries have been tentatively linked to elevated exposure to endocrine disrupting agents. One such agent, bisphenol A (BPA), is a component of plastics and resins that leaches into food and beverage products and has been detected in normal human serum, amniotic fluid, and fetuses. BPA has estrogenic properties both in vitro and in vivo and pre- or perinatal treatment of rodents promotes inappropriate development and proliferation of mammary gland alveoli in adults. While this mammary gland response portends a predilection for development of mammary cancer, no studies have been reported that directly assess whether mammary cancer susceptibility is indeed altered following BPA exposure. Our central hypothesis is that prenatal exposure to bisphenol A will increase mammary gland tumor susceptibility in adult mice. We predict that this increase in susceptibility will be due to combined developmental changes both within the gland and in the hormonal milieu. During formation of the mammary anlagen, an altered estrogenic environment may convey an irreversible adaptive response, making the gland intrinsically more susceptible to the formation of hyperplasia, a preneoplastic state. In addition to altering end organ sensitivity, we expect a central change in the endocrine environment of BPA exposed females. We anticipate that these factors collaborate, creating an altered transcriptome that predisposes the mammary gland to carcinogenic events later in life. We will directly assess the tumor susceptibility of mammary glands that prenatally exposed to BPA using a chemical carcinogen paradigm. We also will determine if chronic hyperplasia induced by BPA is due to intrinsic mammary epithelial changes, hormonal alterations, or both. Lastly, we will characterize changes in the mammary gland transcriptome that integrate these inputs to form a hyperplastic gland. This will build a foundation for future work examining mechanisms by which expression of these genes/pathways is changed by prenatal BPA exposure as well as determining if overt manipulation of these genes alters breast cancer risk.
DESCRIPTION (provided by applicant): The overarching goal of this research is to evaluate how the insecticide carbaryl alters thyroid hormone action, which in turn can influence critical developmental processes. We will use a non-mammalian, amphibian model because 1) amphibians undergo metamorphosis, which is under direct control of thyroid hormones; 2) amphibians are sensitive to environmental contaminants due to permeable eggs, gills, and skin; 3) previous work with amphibians has shown that exposure to the insecticide carbaryl can lead to precocious metamorphosis, which suggests thyroid hormone action is affected by exposure; and 4) the mechanisms whereby thyroid hormones enact their cellular effects are highly conserved in vertebrates, including humans. The proposed research will evaluate the central hypothesis that exposure to a sublethal concentration of an insecticide induces thyroid hormone action, which can negatively affect growth, development, behavior, and survival of individual anurans. To evaluate our hypothesis, we will examine the following specific objectives using molecular techniques, and laboratory and field studies with green frogs, Rana clamitans, reared from hatching through metamorphosis and into the terrestrial environment: (1) Evaluate the effects of exposure to the insecticide carbaryl on thyroid hormone action, and time to metamorphosis, size at metamorphosis, and survival to metamorphosis; (2) Examine how timing of exposure to carbaryl at different stages of development affects thyroid hormone action and incidence of precocious metamorphosis; (3) Determine the magnitude of the effect of carbaryl exposure on thyroid hormone action and metamorphosis under conditions of changing larval density, a stressful natural condition; and (4) Establish how larval exposure to carbaryl will influence later life stages by evaluating terrestrial performance of metamorphosed frogs. All animals may be particularly vulnerable to contaminants when they are undergoing cell proliferation or general remodeling of existing body plans (e.g., embryonic development, metamorphosis, infancy, childhood development, sexual development). Demonstrating that environmentally relevant concentrations of carbaryl can induce thyroid hormone action in both the laboratory and the field would offer powerful evidence to suggest that similar biochemical effects could occur in humans under natural conditions; this research will also indicate how natural environmental stressors may further increase susceptibility to effects of contaminants. The research outlined here will specifically address NIH goals linked to understanding processes related to human growth and development, and the biological effects of environmental contaminants using a non- mammalian model.
Project Narrative: Demonstrating environmentally relevant concentrations of carbaryl can induce thyroid hormone action and that timing of development influences this response in an amphibian model would allow us to link contaminants to irreversible developmental changes that can influence brain development, rates of physical development, behavior, and future growth and survival. This research could suggest that humans may be most negatively affected by contaminant exposure during times of rapid growth and development, especially during embryonic development or onset of sexual maturity. Our proposed research uses an amphibian model and could help explain the role environmental contamination has in diverse phenomena such as thyroid diseases, behavior disorders, and early maturation in humans, all of which are or can be influenced by thyroid hormones.
DESCRIPTION (provided by applicant): Many variant alleles of Brca1 and Brca2 are highly penetrant and predispose carriers to early onset breast cancer. These alleles, however, account for a minority of familial breast cancer cases. It is likely that the majority of such cases are mediated by low penetrance alleles in other genes involved in DNA damage response or DNA repair. Among these is the gene that encodes the cell cycle checkpoint kinase 2 (CHEK2), which is the focus of this application. In particular, we will focus on a CHEK2 (Chk2 in mouse) allele, designated CHEK2*1100delC, for which we have made a knockin mouse model. The CHEK2*1100delC allele, which predisposes to breast cancer in humans, has a single nucleotide deletion near its 3' end and encodes a C-terminal truncated protein lacking much of the kinase domain. Significantly, this allele confers increased breast cancer risk, but not in patients who have variant BRCA1 or BRCA2 alleles, suggesting that CHEK2 participates in the same pathway as the BRCA proteins. The CHEK2 kinase is a participant in a cellular DNA damage response, and interacts with other members of the pathway, including BRCA1 and BRCA2. CHEK2 phosphorylates BRCA1, and we now provide evidence that CHEK2 also phosphorylates BRCA2, a protein involved in recombination-mediated DNA repair. We demonstrate that CHEK2 phosphorylates BRCA2 at residues important for the association of BRCA2 with Rad51 and for the deposition of Rad51 onto single strand DNA, an activity required for efficient recombination-mediated DNA repair. We propose to utilize our Chk2*1100delC mouse knockin model to test the hypothesis that Chk2 kinase activity regulates the function of BRCA2 in recombination-mediated DNA repair, at least in part. Further, we will test whether the absence or reduction in Chk2 kinase activity, and resultant genomic instability, contributes to increased risk of cancer, particularly as a consequence of environmental exposure.
DESCRIPTION (provided by applicant): Altered regulation of immune genes by chemicals or disease states can seriously compromise immune function. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD or dioxin) is a persistent environmental contaminant that produces a variety of toxic effects. Among these effects, alterations in immune function are some of the earliest detected at the lowest exposure levels. There have been more papers written about TCDD's effects on the immune system than about any other environmental hazard, but, despite almost 30 years of research, there is still no clear understanding of TCDD's mechanism of action on any aspect of the immune system. Additionally, TCDD is only one representative of a large class of chemicals that likely constitute a significant hazard to human health. Many of the toxic effects of TCDD and related chemicals have been attributed to activation of the aryl hydrocarbon receptor (AhR) and transcriptional modulation through binding of the AhR to DNA at dioxin responsive elements (DRE). Although there are likely several immune genes modulated by TCDD through an AhR/DRE mechanism, our previous studies have specifically demonstrated TCDD- mediated inhibition of Ig heavy chain gene expression in activated B cells, coupled with TCDD-induced binding of AhR to DRE sites located within the immunoglobulin heavy chain 3' regulatory region (3' IgH RR). With the stated specific aims (SA), we will test the HYPOTHESIS that TCDD inhibits Ig heavy chain gene transcription and class switch recombination through an AhR-dependent modulation of 3'IgH RR activity which involves protein binding to DRE and KB motifs within this regulatory region. SA1: Determine the enhancer regions responsible for LPS-induced activation and TCDD-induced inhibition of 3'IgH RR activity in the context of chromatin. SA2: Determine if AhR and/or NF-icB/Rel proteins are essential to the effects of TCDD on Ig expression and 3'IgH enhancer modulation. SA 3: Determine the effect of TCDD on Ig heavy chain class switch recombination. These studies will contribute to our long-term goal of elucidating the physiological role of the AhR in regulating Ig gene expression. The proposed work is significant in that it will contribute new insights into the molecular basis for the effects of TCDD on B-cell biology. These insights will be important not only to improving our understanding of the human exposure risks to a group of persistent environmental contaminants but they will also likely provide new perspectives on approaches to modulating B-cell function.
DESCRIPTION (provided by applicant):
Exposure Biomarkers of Polyfluoroalkyl Compounds in Persons Living in the Ohio River Valley Polyfluoroalkyl compounds (PFCs) such as perfluorooctanoate (PFOA) and perfluorooctane sulfonate (PFOS) have been detected in humans and wildlife, and are associated with adverse health effects in laboratory animals and humans. Research in animals has noted immunotoxicity in several species, which may be secondary to the activity of PFOA as an agonist to the peroxisome proliferators activated receptor-1 (PRAR-1), although no clinical evidence of immune system perturbation has been noted in humans. In studies of PFC environmental biomarkers in 6-8 year-old girls from Greater Cincinnati (N=60), we found that 38/42 girls living in one school district of Northern Kentucky (NKY) had serum PFOA concentrations that were above the NHANES 2003- 2004 95th percentile value for children 12-19 years (8.6 ng/ml), with a geometric mean of 16.8 ng/ml. The analysis of their residential history data suggested that the exposure was present throughout NKY and not limited to one school district, and that the time of primary exposure was prior to 2002. Length of residence in NKY and being breast fed were significant predictors of serum PFOA. In the proposed study, we will (Aims 1 and 2) sample serum from 510 persons throughout the Ohio River Valley, using the river as a drinking water source, and in areas served by two different water treatment systems, to determine the prevalence of serum PFOA concentration above the NHANES 95th percentile, (Aim 3) use information about the geographical distribution and residential history of those with elevated concentrations to determine the existence and strength of modifiers of serum PFOA concentrations, in those of different ages, gender, pregnancy status, and history of being breast fed, and (Aim 4) determine if those with the highest serum PFOA or PFOS concentrations exhibit evidence of an inflammatory response, using molecular markers of interleukins and C-reactive protein, or if serum PFOA or PFOS concentration is related to body mass index.
DESCRIPTION (provided by applicant): Trichloroacetate (TCA) and dichloroacetate (DCA) are by-products found in the chlorinated drinking water. The compounds are found to produce liver toxicity and cancer, in animals. We propose that long term exposure to TCA and DCA induces oxidative stress (OS) in mice. These mechanisms occur earlier than production of any detectable toxic, or carcinogenic effects in the liver. The effects are also produced in response to much lower doses than those required to produce detectable liver toxicity or cancer. We also propose that long term exposure to mixtures of DCA and TCA, produces more effects than those produced by the individual compounds. Also, marginal vitamin E deficiency increases the toxic/carcinogenic risks of the compounds. Groups of male B6C3F1 mice will be treated post orally, with daily doses of TCA and DCA, for 4-30 weeks. At the end of the treatment periods, mice will be sacrificed and phagocytic cells will be collected and assayed for production of reactive oxygen species (ROS) and tumor necrosis factor-alpha (TNF-alpha). Livers will be collected and investigated for development of histopathological changes, as well as for the determination of various biomarkers of OS, including ROS, lipid peroxidation, DNA damage, glutathione level, and activities of different antioxidant enzymes. The doses of TCA and DCA and times required to produce significant changes in the biomarkers of OS, will be determined and compared with those required to produce detectable liver toxicity or cancer in the same animals. Human risk due to exposure to toxic chemicals is, in general, based on the doses and times required to produce cancer in rodents. This project will identify markers of toxicity/carcinogenicity that are significantly changed in response to TCA and DCA. These changes occur at doses and times that are much less than those required to produce detectable cancer. Hence cancer production will underestimate humans risk when used as an end point, while that risk will not be underestimated if based on induction of markers of OS as proposed in this project. Furthermore, the results can be used as basis for studies on human populations to determine the association between changes in biomarkers of OS and consumption of the chlorinated drinking water, as well as with the risk of that human population for cancer development. The study will also be the first to determine the possible increases in effects when TCA and DCA exist in the form of mixtures. This is important since humans are exposed to mixtures of the compounds in the drinking water, rather than to the individual compounds. Results from studies on the mixtures can be used as bases for adjustment of the allowable levels of the compounds in the chlorinated drinking water. The study will be also the first to determine any possible increases in the toxic/carcinogenic risks of the compounds, associated with marginal vitamin E deficiency. Dichloroacetate (DCA) and trichloroacetate (TCA) are toxic and carcinogenic by-products formed during the process of drinking water chlorination. The present study will determine indicators for the toxic effects of the compounds that can be detected much earlier than cancer production in animals. These can be used as basis to determine similar indicators for human risks from exposure to the compounds in the drinking water, and also to adjust the allowable amounts of the compounds in the drinking water.
Crisp Terms/Key Words: environmental exposure, oxidative stress, water supply, water pollution, environmental toxicology, longitudinal animal study, free radical oxygen, vitamin E deficiency, cancer risk, liver neoplasm, environment related neoplasm /cancer, liver toxic disorder, tumor necrosis factor alpha, histopathology, chlorination, dichloroacetate, acetate, disease /disorder proneness /risk, biomarker, laboratory mouse
DESCRIPTION (provided by applicant): This competing renewal application aims at investigating the hypothesis that exposure of human melanocytes to ultraviolet radiation (UV) and/or the physiological agonist 1-melanocyte stimulating hormone (1-MSH) or antagonist agouti signaling protein (ASIP) modulates the expression of the melanocortin 1 receptor (MC1R) gene, and regulates the activation of the receptor by affecting its desensitization, internalization and resensitization. The MC1R is a Gs protein-coupled receptor with seven transmembrane domains that is expressed on human melanocytes. Activation of this receptor by its agonists 1-MSH or ACTH stimulates cAMP formation and the synthesis of the brown-black eumelanin, which confers cutaneous photoprotection. We have shown that activation of the MC1R is pivotal for the UV-induced tanning response, and importantly, reduces the extent of UV-induced DNA damage by enhancing nucleotide excision repair and counteracting oxidative stress in human melanocytes. These effects explain why loss-of-function alleles of the MC1R are associated with increased risk for melanoma. We are proposing that MC1R expression and function are regulated at different levels in response to UV, its physiological agonists and antagonist. To investigate the above stated hypothesis, we propose the following three Specific Aims. First, we will investigate the regulation of MC1R gene expression and receptor trafficking by real time RT-PCR, Western blotting, and immunostaining. Second, we will determine the activation of the MC1R, by quantitating the number of membrane receptors/melanocyte, its agonist-induced desensitization, internalization, and resensitization. Third, we will define the roles of G protein receptor kinases (GRKs) and 2-arrestins in MC1R surface expression and sequestration. The significance of the proposed studies lies in the critical role of the MC1R and melanocortins in the UV responses of human melanocytes, and in filling the gap in the existing knowledge about regulation of this receptor in the physiologically-relevant cell, the epidermal melanocyte. Given that the MC1R is a melanoma susceptibility gene and an important determinant of the UV-induced tanning response, elucidating the regulation of MC1R expression and activation will lead to new strategies to prevent melanoma and other types of skin cancer by increasing the activity of the MC1R and optimizing the photoprotective capacity of the melanocyte, particularly in high risk individuals. PUBLIC HEALTH RELEVANCE The outcome of this grant application is expected to lead to new strategies for prevention of melanoma, the deadliest form of skin cancer, and of non-melanoma skin cancers. These strategies will be based on modulating the activity of the melanocortin 1 receptor by mechanisms that will be elucidated during the course of this grant proposal. The incidence of melanoma in the U.S. and Eastern countries continues to rise with no effective treatment for advanced disease; hence the relevance of this project for public health.
DESCRIPTION (provided by applicant): The objectives of this grant application are to continue and expand research directed towards the full characterization, total synthesis, and environmental detection of the azaspiracid natural products. The azaspiracids are marine toxins that accumulate in shellfish cultivated and collected for human consumption. Azaspiracids are likely to be produced by the dinoflagellate Protoperidinium crassipes, that has widespread geographical distribution. The toxins have a unique mode of action, are neurotoxic, necrotic, and tumor promoting in mice. They have caused numerous human poisonings via the consumption of contaminated shellfish, and their surveillance and quantitative detection is critical to minimize further human exposures. The structures of the azaspiracids are complex and only partially defined. They are amino acids of enormous proportion, having 40 contiguous carbons in their backbone and containing an unprecedented array of structural features. These include THF-fused trioxadispiroketal moiety and a polycyclic spiroaminal terminus. The specific aims of this proposal are to develop efficient syntheses of both enantiomers of the C26-C40 spiroaminal-containing domain, develop enantioselective syntheses of the the trioxadispiroketal-containing C1-C20 domain, develop standards and haptens for the sensitive and quantitative environmental monitoring of the azaspiracids, completely define the stereochemistry of azaspiracid-1, and develop an enantioselective total laboratory synthesis of azaspiracid-1. This work will continue to contribute to understanding of the structure, occurrence, toxicology, and chemical and biological reactivity of the azaspiracids. It will provide continued impetus for fundamental developments in organic synthesis, including novel strategies and tactics for the total synthesis of complex molecular architecture. A novel immunostereochemical study is proposed to aid in the complete structural definition of the azaspiracids. This approach fuses synthetic organic, natural products, and immunochemistry to support structural elucidation and the development of an ELISA system for the universal detection of all of the azaspiracids that are currently known.
DESCRIPTION (provided by applicant): The broad long-term overall goal of the proposed research is to understand the physiological role of estrogen (E2) and estrogen receptors (ERs) during neuronal development and to determine to what extent environmental estrogens (EEs) can mimic or inhibit the effects of E2 in these developing neurons. Mechanisms of rapid E2-mediated signal transduction are not well understood. These actions vary greatly from cell type to cell type, and may even vary in the same cell. In addition to reproductive tissues & breast cancers, cells of the nervous system are a clinically very important E2 targets. Environmental estrogens, also known as endocrine disrupting chemicals, are a diverse group of compounds that can mimic or antagonize the normal actions of E2. The extent to which EEs impact the developing nervous system is unclear. Proposed experiments using primary cultures of estrogen-sensitive neonatal rat cerebellar neurons and the developing cerebellum as non-sexually related neuronal models, will address the following Specific Aims - Specific Aim 1 is to determine whether or not structurally diverse EEs modulate ERK1/2 signaling in developing cerebellar neurons. It is hypothesized that in E2-responsive neurons, EEs rapidly modulate ERK1/2 signaling and neuronal physiology in an E2-like fashion, and that differences in the chemical structure of individual EEs determine their ability to activate ERK-signaling &/or antagonize the rapid actions of E2. Specific Aim 2 is to determine the nature of the signaling mechanism underlying E2-mediated rapid activation of ERK-signaling. It is hypothesized that a plasma membrane, localized version of ERp is acting as the mediator of rapid E2/EE-induced ERKsignaling. Membrane E2-binding proteins from primary granule cell and hippocampal neuronal cultures will be affinity purified and identified through LC-tandem mass spectrometry. Immunocytochemical and loss-of-function experiments are proposed to demonstrate the function of identified candidate membrane ERs. The ability of ERa, ERp, and the orphan G-protein coupled receptor GPR30 to act as the membrane ER will be directly addressed.
Learning and behavior problems among US children appear to be rising steadily, however the etiology for
these behavioral changes is unknown. Exposure to low-levels of various neurotoxins has been suggested
as a potential factor in these changes. For example, low levels of lead are known to be associated with
learning deficits and social behavior problems. One issue with animal models for the effects of various
neurotoxins is that they are limited to investigating only one factor even though human exposures occur
together with many other environmental factors. For example, it is known that there is a differential exposure
to heavy metals in low social economic status households. Therefore, we will investigate the effects of lead
and manganese (since manganese is known to potentiate the increase in the amount of lead that
accumulates in the brain) in combination with stressors (isolation, overcrowding, or limited resources with
maternal depression) to mimic human conditions. The first aim is to determine which stressor has the
greatest impact on heavy metal exposure by measuring physiological parameters during the period of
administration as well as investigating the long-term effects of this combination on neurotransmitters and
long-term potenitation induction. The second aim is to investigate the dose-dependency of lead or
manganese in combination with stress on the learning ability and social aspects of the animals. We will be
using a novel approach to investigating learning and memory in these animals by using a combination of
learning tests that test different types of learning. This is relevant to how humans would have to learn as
well. This aim will provide a foundation to model low level exposure and to understand the functional
changes that occur. The third aim is to combine lead, manganese and stress and assess the impact of this
more relevant combination to human exposures on learning and memory and social behavior. The last aim
is designed to investigate the developmental changes that occur related to the three combination exposure
by using MRI as well as looking at the nuerophysiological response when the animals are adults. These
studies will provide much needed data on the low-level effects of neurotoxins in combination with
environmentally relevant stressors. Potential mechanisms will be explored by investigating neurotransmitter
and neuroendocrine systems and the glutamate receptor complex.
DESCRIPTION (provided by applicant): The maturation of the central nervous system requires a complex sequence of processes over an extended period of time, making this organ particularly vulnerable to environmental influences. The long term behavioral morbidities associated with prenatal and postnatal exposures to environmental chemicals remain uncertain. We also lack data on direct anatomical evidence of neurological damage resulting from lower level exposures to known developmental neurotoxicants. We have very little information on how genotypic variances may function as exacerbating or protective factors in neurodevelopmental morbidity. In the area of children's environmental health there has never been a systematic attempt to 1) Determine the impact of early exposure on behavior in later adulthood; 2) Identify the specific neuroanatomical substrates and physiological processes that may have sustained damage as well as their neuropsychological correlates in behavior; and 3) Assess interactions between genes encoding proteins involved in neurotransmitter molecular functions and environmental exposures in the expression of neuroanatomical anomalies, and behavioral problems. In this study, we will examine the relationship between prenatal and postnatal exposure to lead (Pb), concurrent exposure to manganese and neuroanatomical anomalies measured non- invasively using magnetic resonance imaging the spectroscopy (MRI/MRS), adult attention deficit hyperactivity disorder (A-ADHD) and related co-morbidities as well as criminal behavior in subjects in their mid 20s. Another goal of this study is to examine these dose-response relationships in the context of genotypic variances related to dopaminergic and serotonergic function in the central nervous system. The proposed research builds on a longstanding prospective study of subjects recruited prenatally beginning in 1979 who continue to be followed today. The Cincinnati Lead Study (CIS) cohort is predominantly African- American with a history of socioeconomic disadvantages. Out of an initial cohort of 305 subjects we continue to track approximately 250 in their early 20s. Exposure to Pb has been assessed serially in blood from the first trimester to 17 years of age. We will be assessing concurrent Mn exposure in blood as well as in brain using an innovative MRI-derived biomarker as a possible modifier of lead's long lasting effects on neurobehavior. There is a rich database covering the cohort's lifespan medical and social histories.
Crisp Terms/Key Words: environmental exposure, clinical research, gene environment interaction, comorbidity, low socioeconomic status, criminal behavior, interpersonal relations, mental disorder diagnosis, neuropsychology, early experience, aggression, magnetic resonance imaging, employment /unemployment, developmental neurobiology, neuroanatomy, lead poisoning, manganese, neural information processing, human subject, genetic polymorphism, dosage, attention deficit disorder, brain, adult human (21+)
DESCRIPTION (provided by applicant): Methylmercury (MeHg) is a well-recognized neurotoxic agent that can affect the fetus. In the tragic disasters that took place at Minamata, Japan, and subsequently in Iraq, many infants were exposed transplacentally to MeHg. The victims manifested a cerebral palsy-like syndrome characterized by severe mental retardation, motor dysfunction, and repeated convulsions. Histological examination revealed widespread neuronal degeneration, abnormal arrangement of neurons in the cerebrum and cerebellum, and diffuse white matter gliosis in the brain of victims. Even now, MeHg is a major environmental problem, since it accumulates in the aquatic food chain and consequently creates a risk to the human brain, especially that of the fetus. The long-term goal of the present proposal is to elucidate the cellular and molecular mechanisms underlying MeHg-induced malformation of the brain. We will focus on the effects of MeHg on neuronal cell migration in the developing brain since it has been suggested that MeHg can disturb the process of neuronal cell migration. To this end, we will use the cerebellum as a model system because the effect of methylmercury on brain growth is marked in the cerebellum. We will determine the effects of MeHg on the migration of cerebellar neurons. First, we will determine how MeHg administration affects the migration in vivo and in vitro. Second, we will determine if MeHg alter migration by altering the Ca2+ signaling pathway. Third, we will determine if the interaction between the Ca2+, cAMP and cGMP signaling pathways synergistically alters the effects of MeHg on migration. Fourth, we will determine if the interaction between the Ca2+ signaling and the ROS production plays a crucial role in MeHg-induced impairment of migration. The fundamental mechanisms whereby MeHg administration leads to the disturbances of brain development have not been delineated definitively. Answers to the questions raised in this project will provide a new understanding as to how prenatal and early postnatal exposure to MeHg causes malformation of the brain. Lay Summary PUBLIC HEALTH RELEVANCE In the late 1950s, many infants born in Minamata of Japan developed characteristic neurological symptoms, including mental retardation and cerebral palsy. Later it has revealed that methylmercury (MeHg) induced congenital intoxication via the transplacental transfer of mercury to the fetus. The term fetal Minamata disease (FMD) was used to describe such medical phenomenon. The most characteristic symptoms of FMD are mental retardation, dysarthria, cerebellar symptoms (such as ataxia), and deformity of the limbs. Furthermore, histological studies revealed that in FMD disruption of the cerebral and cerebellar cytoarchitecture was prominent. Many neurons appeared to be hypoplastic, ectopic, dysplastic, and disoriented, strongly indicative of disrupted migration, maturation, and growth. Extensive neuronal loss occurred throughout the brain. MeHg exposure remains a major public health concern because of natural and anthropogenic release of inorganic mercury into the aquatic environment, where it is biotransformed by algae and bacteria into MeHg. This can pass along the food chain and, eventually, to man. The long-term goal of the present proposal is to reveal the cellular and molecular mechanisms underlying the MeHg-induced malformation of the brain. In particular, we focus on the study examining whether MeHg exposure impairs neuronal cell migration in the developing brain. The fundamental mechanisms whereby MeHg exposure leads to disturbances of brain development have not been delineated definitively. Answers to the questions raised in this project will provide insights for development of therapies that counter MeHg-induced brain malformation.
DESCRIPTION (provided by applicant): There is strong evidence that diesel exhaust particles (DEP) can augment sensitization to aeroallergens and enhance expression of Th2 cytokines and allergy symptoms. Hence, DEP exposure early in life may increase allergic diseases in young children, and this risk may be modified by genotype. The purpose of this renewal is to elucidate the independent and/or combined contributions of DEP, aeroallergens, genetics, and other factors associated with allergic disease for children ages 1-4. This proposal is a request to finish testing the children at age 4 who have been followed prospectively from birth. We also propose to optimize the analysis of previously collected air samples for estimates of DEP. This study has thus far evaluated 758 infants from atopic families. Through age 3, we have 88% cohort retention, and of these, 87% are in complete study compliance. Infants received annual medical exams and had yearly skin prick tests (SPT) for 15 aeroallergens through age 3. We have collected DNA samples from children and parents and collected hair samples from infants to assess nicotine and cotinine. We established a network of 18 air sampling stations for PM2.5 and obtained five years of exposure estimates for DEP. To-date, through 28 publications, we have demonstrated a significant association with the independent or combined exposures to DEP, aeroallergen and tobacco smoke (ETS) and infant sensitization, allergic rhinitis and/or wheeze. These findings have been used by the U.S. Senate in support of the Diesel Emission Reduction Act of 2005. Further, we have findings impacting clinical evaluations as no other study has tested 15 aeroallergens in infancy. We found that at age 1 and 2, 18% and 36% of infants, respectively, are SPT+ to aeroallergens. Also, our results show significant gene:environment interactions. For example, infants with the IV/W genotype for GSTP1 and the highest DEP exposure (>0.5 A/g/m3) were significantly more likely to wheeze (18% vs 38%, p<0.01). Also, we showed that for African-Americans with high ETS exposure and the CT/TT genotype for IL4 C-589T, there was a ten fold increased risk of wheezing. In summary, this study has the potential for making a significant public health impact as allergic diseases are the most common chronic diseases of childhood. This research is poised to make additional innovative contributions to aid in the reduction of childhood allergic morbidity and mortality related to common environmental exposures.
DESCRIPTION (provided by applicant):
Environmental exposure to fundamentally diverse agonists such as bacteria and air pollutants can signal through shared mechanisms to activate the innate immune system. Although the roles of pulmonary lymphocytes in response to bacterial infection are well established, the roles of pulmonary lymphocytes in response to air pollutants are unclear. We propose to explore the paradigm that the same lymphocyte effector functions necessary to protect the lung from pathogens can lead to chronic pulmonary disease when stimulated by persistent exposure to environmental toxicants. To understand shared roles and mechanisms of lymphocyte activation following bacterial infection and toxicant exposure, we will study epithelial cell lymphocyte interactions in response to the common bacteria Pseudomonas aeruginosa (PA) and the ubiquitous air pollutant, acrolein. PA is a well-characterized gram-negative bacterium that constitutes a major cause of nosocomial infections. Acrolein is a hazardous air pollutant found in tobacco smoke, photochemical smog and diesel exhaust. We have examined specific mediators involved in the signaling of epithelial cell stress to the pulmonary immune system and identified NKG2D receptor activation as a potential mechanistic link between epithelial cell stress and lymphocyte activation. The central hypothesis of this application is that chronic acrolein exposure induces NKG2D ligand expression on pulmonary epithelial cells, activates cytotoxic lymphocytes that contribute to the development of Chronic Obstructive Pulmonary Disease (COPD). The Specific Aims are to (i) determine the role of NKG2D receptor activation in the clearance of pulmonary PA infection, (ii) determine the role of NKG2D receptor activation in the development of acrolein-induced airspace enlargement, and (iii) define the consequences of conditional NKG2D ligand expression on pulmonary epithelial cells. These shared mechanisms represent potential disease points that may shift the immune response from protective to pathological. Further, the development of the novel conditional NKG2D ligand-expressing transgenic mouse model will allow us to dissect the mechanisms of epithelial cell-lymphocyte interactions in emphysema. The successful completion of these studies will provide a better understanding of the pathways and mechanisms of immune system activation in response to environmental exposures that contribute to the pathophysiology of chronic airway diseases.
DESCRIPTION (provided by applicant)
Asthma and respiratory infection pose major burdens to child health. Prenatal tobacco exposure is a key risk factor for wheeze in young children, but there is little data about the relationship of prenatal low level exposures to tobacco (e.g. passive exposure of nonsmoking pregnant women) with risk of childhood wheeze or respiratory infection. There is emerging, yet limited evidence for novel environmental risk factors for wheeze such as phthalates. Advances in biomarkers offer an ability to quantify indicators of internal dose directly, reduce exposure misclassification, and enhance our ability to link exposures with disease. The primary objective for this K23 proposal is to rigorously characterize the relationship of common environmental exposures with risk of wheeze and respiratory infection in early childhood using serial biomarkers of exposure. The specific aims are to: 1) Test which measure of prenatal tobacco smoke exposure (different biomarkers, parent-report) is most strongly associated with risk of wheeze, respiratory infection, and pulmonary inflammation (fraction of exhaled nitric oxide, FeNO) in the first three years of life; 2) Investigate the association of low levels of maternal passive tobacco smoke exposure during pregnancy with risk of wheeze, respiratory infection, and pulmonary inflammation (FeNO) in children of nonsmoking mothers in the first three years of life; and 3) Investigate the association of prenatal phthalate exposure with risk of wheeze, respiratory infection, and pulmonary inflammation in the first three years of life. This proposal is novel because it uses serial biomarkers to measure individual exposure burden and low levels of exposure objectively throughout pregnancy and early childhood. Additionally, we will be the first to explore the role of prenatal phthalate exposure with the risk of childhood wheeze, respiratory infection, and pulmonary inflammation using biomarkers. Through this K23, the applicant will develop expertise in pediatric environmental epidemiology to position him to help broaden the model of environmental toxicology and respiratory epidemiology in children. It will also provide the background and training needed to develop an independent research career in clinical pediatrics and enhance our understanding of how environmental risk factors affect the development and exacerbation of pediatric asthma and respiratory disease and integrate this knowledge into prevention and treatment efforts.
DESCRIPTION (provided by applicant): This project is concerned with the dispersal of fungal spores in the indoor environment. At a time of widespread concern about microbial contamination of buildings, this project is motivated by the following questions: What mechanisms cause and maximize spore release in indoor fungi and what practices might help to minimize spore release? There are two specific experimental aims. Aim 1 is designed to elucidate mechanisms of spore release in indoor fungi. Despite the ubiquity of other fungi in the indoor environment, and their proven allergenic potential, we know little about their mechanisms of spore release. A combination of video microscopy and biomechanical techniques will be used to understand how the commonest indoor molds (sexual ascomycetes), sexual ascomycetes (that release ascospores), and basidiomycetes achieve spore release. Building upon the information gathered through the experiments of Aim 1, the experiments of Aim 2 will explore the environmental conditions (particularly humidity and airflow) that control spore release for each category of release mechanism. Fungal proliferation is almost inevitable following water intrusion into buildings, but unless spores become airborne, contamination will have little effect upon air quality. This means that controlling spore release may be an effective way to maintain air quality even in cases of severe building contamination. Furthermore, by focusing upon spore release rather than fungal growth it may be possible to reduce the use of potentially-hazardous chemicals during mold remediation efforts.
DESCRIPTION (provided by applicant): Project Summary: The lack of proper biomarkers has greatly hampered investigations of the human health effects of the toxigenic fungus, Stachybotrys. Attempts to relate inhalation exposure to pulmonary, immune and neurological toxicities have had only circumstantial evidence supporting the apparent exposure. Epidemiological and pathophysiological studies would be greatly facilitated by the development of biomarkers that can be quantitatively related to the dose and timing of the exposure. We have recently found that purified satratoxin G, a macrocyclic trichothecene from Stachybotrys with 2 epoxides, forms stable adducts with human serum albumin. This offers the potential for quantitative biomarker 'dosimeters' in the form of protein adducts and related urinary metabolites in parallel to that used for the epoxide derivative of the mycotoxin, aflatoxin B1. Hypothesis: Inhalation exposure of humans to strains of Stachybotrys chartarum producing satratoxin G will result in the formation of covalent adducts of serum albumin and/or hemoglobin in sufficient quantities to be measured using mass spectrometry or radioimmune assay. This project proposes to: 1) Complete the identification of the amino acyl satratoxin adducts of recombinant human serum albumin, determine the chemical structure of the adducts, and ascertain the relative reactivity of the amino acyl residues being modified. 2) Confirm the presence of these adducts and/or metabolites in samples of biological materials including serum, red blood cells and urine from exposed rats and humans. 3) Develop practical quantitative biomarker assays for satratoxin-albumin and hemoglobin adducts using exhaustive proteolysis and immunoaffinity chromatography with analysis by mass spectrometry and by radioimmune assay. Similar mass spectrometry assays will be developed for related urinary metabolites. 4) Develop parallel assays for macrocyclic trichothecenes in environmental dust samples. Relevance: Over the past decade there has been increasing public concern about the health effects of "toxic mold" or "black mold" (i.e. Stachybotrys) in home and work environments. The lack of objective tools to relate the amount of exposure to the observed health problems has hampered attempts to clearly demonstrate such mold-related health effects. This project proposes to develop quantitative assays for such investigations.
DESCRIPTION (provided by applicant): The aryl hydrocarbon receptor (AHR) is a ligand activated transcription factor that mediates the biological and toxicological effects of a broad range of structurally diverse chemicals, including 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD). Inherent properties of the AHR signaling pathway, including AHR expression levels and the affinity of AHR for specific ligands, can underlie large variations in the relative potency of different ligands and the sensitivity of different animal groups. However, neither the exact mechanisms by which AHR activity leads to toxicity nor the endogenous, non-toxicological functions of AHR signaling are well understood. Relative to most other vertebrates, frogs are extremely insensitive to TCDD toxicity. Our group has identified two AHRs from the African clawed frog (Xenopus laevis), recently duplicated paralogs called AHR1? and AHR1?. Both proteins bind TCDD with at least 25-fold lower affinity than the AHR from a highly sensitive strain of mouse, likely accounting for the dioxin-insensitive phenotype. The proposed project will take advantage of the unique amino acid sequence, functional properties, and phylogenetic position of the frog AHRs to probe their structural interactions with a range of xenobiotic and naturally occurring ligands and to contrast their function with AHRs from TCDD-sensitive species and with each other. We propose three specific aims: (1) Using site-directed mutagenesis to make the frog AHRs more "mouse-like," we will test the hypothesis that changes in one or a few amino acids within the putative ligand binding domain confer low TCDD affinity. This comparative approach will contribute significantly to the identification of important structural features of AHR's ligand binding pocket. (2) We will determine the relative potency of structurally diverse candidate ligands. Although X. laevis AHRs bind TCDD with low affinity, they may remain highly responsive to structurally distinct compounds, especially putative endogenous ligands. We will test this hypothesis by establishing structure-activity relationships for a range of candidate ligands, including indole-containing compounds that bind mammalian AHRs. (3) We will determine the functional differences between AHR1? and AHR1?, examining expression patterns, enhancer preferences, and broad-based changes in gene expression mediated by individual paralogs. These studies will test the hypothesis that the AHR paralogs exhibit distinct functions, possibly partitioning multiple roles of the single mammalian AHR. Overall, this comparative approach in a novel model system will provide important basic information about the structure and function of all vertebrate AHRs. Understanding the differences between frog and human AHRs will also aid risk assessment by refining interpretation of toxicological data derived from FETAX (Frog Embryo Teratogenesis Assay-Xenopus) and similar developmental toxicity tests that employ frog embryos. This project will study the aryl hydrocarbon receptor (AHR), a protein that mediates the toxic effects of environmental contaminants such as dioxin. We will compare the structure and function of the AHR from frogs, which are insensitive to dioxin toxicity, with AHRs from more sensitive animals like mice and humans. This comparative research will help scientists understand important differences between frogs and humans as they interpret toxicology studies using frog embryos as a model system for measuring the effects of chemicals and environmental samples. It will also provide basic information about the original, non-toxicological function of the AHR.
DESCRIPTION (provided by applicant):
Particulate matter (PM) comprises a significant portion of air pollution. Particles in the range of 0.01 to 1.0 micron (PM1.0) are of particular concern, as these deposit in the lung exponentially with decreasing size. Within the alveoli, PM has the potential to initiate inflammatory and immune responses. Particles <1.0 micron are a heterogeneous mixture based on source generation and composition. Studies have shown that particle number, irrespective of composition is critical for macrophage-mediated alveolar clearance due to an "avalanche" effect. Understanding the potential impact or particles on human health is hampered by our ability to accurately characterize average, peak, and cumulative personal exposures in population studies. The proposed study will develop a miniature personal sensor for the detection of PM1.0 that will be field worthy and child friendly. The resulting will enable numerous applications by providing an integrated cyclone, saturator, condenser, and optical detector package consuming only milliwatts of electrical power, and functioning independent of physical orientation and a normal child's activity. The sensor will also have wireless function and position tagging using the advanced information transfer technology developed by NASA, Thus, this study is unique due to the miniature size of the sensor package allowing for constant wear during the daily activities of young children, the ability to monitor PM1.0, and the incorporation of the GPS device which will enable the location of specific exposures to be recorded. The need for personal monitors capable of measuring micro-environmental PM exposure is especially relevant where exposure to many pollutants is highly variable often across short distances, particularly in urban areas, and dependent upon personal activity patterns.
DESCRIPTION (provided by applicant): We believe that unique volatile signatures are associated with fungal outbreaks associated with production of aflatoxin. The long-term goal of the project is to demonstrate the feasibility of our innovative nano-crystalline tin oxide sensor detecting these unique Microbial Volatile Organic Compounds (MVOCs) at very low levels and differentiating between low levels of these unique organic vapors and other Volatile Organic Compounds and MVOC's. The resulting detector would be set inside a grain storage container to protect food safety. The first aim in this research is to identify a catalyst and operating condition, which allow these unique volatiles to be selectively detected in crop enclosures containing both the unique volatiles and potential identified interferants. Combinatorial chemistry methodology will be used to establish the conditions for unique volatile selectivity. Secondly, our aim is to provide baseline information, under biologically relevant conditions to substantiate the use of particular volatiles as markers of aflatoxin contamination. This will be accomplished by carrying out research on host plant-fungus interactions involved in the aflatoxin contamination process and doing volatile analysis on different fungal strains. The conductance response of the sensor is proportional to the concentration of these gases; that is, the higher the concentration of the gas, the greater the response. At the conclusion of this project, we expect to have demonstrated proof of principle for a sensor capable of selectively determining concentrations of unique mold related volatiles as low as 1 ppb in the presence of other typical crop related volatiles. Aflatoxins are toxic metabolites produced by certain molds in/on foods and feeds. The substance is so toxic that it is one of 19 contaminants-along with mercury and DDT-for which the U.S. Food and Drug Administration imposes strict tolerance levels in food, with only trace amounts allowed. Aflatoxins have been associated with various diseases, such as aflatoxicosis, in livestock, domestic animals and humans throughout the world. Aflatoxins have received greater attention than any other mycotoxins because of their demonstrated potent carcinogenic effect in susceptible laboratory animals and their acute toxicological effects in humans. The greatest threat of health hazards to humans come from long term exposures of tainted food products, either from spoilage or from consuming milk or meat from animals that have been fed contaminated feed. The USDA noted that countries that lack the means to regulate and monitor the presence of aflatoxin leave their inhabitants open to unknown poisonings that can continue over long periods of time. Throughout the world, approximately 4.5 billion persons are chronically exposed to largely uncontrolled amounts of the toxin where the toxic affect of aflatoxin on immunity and nutrition combine to negatively affect health factors.
Mycotoxins are carcinogenic compounds that are products of the mold Aspergillus. They affect up to 30% of the worlds food supply and the resulting conclusion is that approximately 4.5 billion persons worldwide are chronically exposed to largely uncontrolled amounts of the toxin. No animal species including humans are immune to aflatoxin poisoning with the greatest threat of health hazards to humans coming from long-term exposure of tainted food products, either from spoilage or from consuming milk or meat from animals that have been fed contaminated feed.
DESCRIPTION (provided by applicant): The long-range goal of this research is to develop an understanding of the mechanisms underlying the adverse health effects and toxicity resulting from exposure to complex mixtures of polycyclic aromatic hydrocarbons (PAH) and chromium, a carcinogenic metal often found as an environmental co-contaminant with PAHs. The objectives of this research project are, (1) to elucidate the mechanisms by which chromium affects inducible gene expression, and (2), to evaluate the effect of mixtures of benzo[a]pyrene (B[a]P), a prototypical PAH, and chromium on the post-translational modifications of chromatin remodeling proteins associated with epigenetic modulation of gene expression. Chromium exposure has been shown to alter inducible gene expression, to form chromium-DNA adducts and chromium-DNA cross-links, and to disrupt transcriptional activator/coactivator complexes. During the previous 4 years of this grant we have shown that chromium blocks gene expression by interfering with the assembly of productive transcriptional complexes at the promoters of inducible genes. We studied the effects of chromium on the expression of genes induced by B[a]P and showed that chromium disrupted the transcriptional regulation of phase I and phase II detoxification genes induced by B[a]P-dependent Ah receptor activation and the inducible expression of over 50 different genes involved in a variety of signal transduction pathways. These effects of chromium were the result of the inhibition of critical chromatin remodeling steps necessary for gene transactivation. Given the central role of histones in maintaining chromatin structure, for the next 5 years of this grant application we propose to test the hypothesis that exposure to mixtures of chromium and B[a]P causes specific histone modifications that generate transcriptionally inactive chromatin and induce non-additive gene expression effects that cannot be predicted from the individual effect of each mixture component. Results from this work will help to generate an understanding of the risks arising from exposure to chemical mixtures and to develop effective means to predict their health effects.
DESCRIPTION (provided by applicant)
Cutaneous melanocytes provide the major physiologic defense against the DNA damaging effects of ultraviolet (UV) radiation by synthesizing and distributing melanin to surrounding cells. Exposure to UV radiation is highly correlated with the transformation of normal melanocytes into melanoma. The mechanisms regulating the response to UV radiation and the steps leading to the oncogenic state have not been elucidated. A hallmark of exposure to UV radiation is an increase in pigmentation by enhanced transcription of genes required for melanin synthesis. Cells also respond to UV radiation by activation of pathways leading to cell cycle arrest, DNA repair, and apoptosis. To understand how a melanocyte responds to UV radiation, it is necessary to characterize the molecular mechanism involved. Activation of gene expression often involves alterations in chromatin structure to make it more accessible to the transcriptional machinery. SWI/SNF enzymes are multisubunit complexes that utilize the hydrolysis of ATP to remodel chromatin structure and facilitate transcription factor binding. They act as tumor suppressors, and are involved in gene regulation and cell cycle control.
Preliminary evidence suggests that SWI/SNF enzymes are required for the activation of melanocyte specific genes (tyrosinase, trp1, and trp2) and for genes regulating cell cycle arrest (p21) and apoptosis (Bcl2). This proposal seeks to investigate the role that SWI/SNF enzymes play in the response to UV radiation. Specific Aim 1 will determine whether SWI/SNF enzymes are required for the enhanced expression of tyrosinase, trp1, and trp2 and for the modulation of other genes upon exposure to UV radiation. Specific Aim 2 will identify the mechanisms by which UV radiation results in activation of a melanocyte specific gene tyrosinase, by looking at changes in chromatin structure and transcription factor binding. Specific Aim 3 will determine a role for SWI/SNF enzymes in cell cycle control.
DESCRIPTION (provided by applicant)
Epidemiological studies have demonstrated an association between fine particulate matter (PM2.5) and acute cardiovascular events. While the precise mechanism(s) that result in transduction of responses to inhaled PM2.5 in the vasculature are currently not well understood, it is clear that these responses can occur within a few weeks of exposure. To investigate the mechanisms by which PM2.5may modulate inflammation and vascular tone with short-term exposure, the researchers will investigate these responses in relevant animal models using real world ambient PM2.5 exposure. Accordingly, they hypothesize that exposure to ambient PM2.5 for 8 weeks induces systemic microvascular inflammation and dysfunction through monocyte/macrophage NAD(P)H dependent reactive oxygen species (ROS) generation and that endothelial nitric oxide plays a pivotal role in modulation of these responses. The investigators plan to examine this hypothesis systematically in appropriate animal model systems that will include genetically modified and tissue-specific conditional gene knockout mice via Cre-lox technology using a versatile ambient exposure system. In the first specific aim, they will investigate the effect of 8-week exposure to PM2.5 in FVBN mice and endothelial nitric oxide synthase (eNOS) knockout mice and examine leukocyte and vascular responses and superoxide production in cremasteric microcirculation. In the second specific aim, the investigators will determine the activation of monocyte/macrophage NAD(P)H oxidase in mice expressing enhanced yellow fluorescent protein (YFP) under the control of c-fms promoter after PM2.5 exposure by flow cytometry, intravital microscopy, and real time PCR. In the final specific aim, they will investigate the effect of PM2.5 exposure on NAD(P)H oxidase derived ROS-mediated monocyte response in the microcirculation in the mice deficient in a critical cytosolic component of NAD(P)H oxidase (p47phox-/- or gp91phox-/-) and tissue specific (endothelium or monocyte/macrophage lineage), conditional gene knockout mice models. Principal component analysis of PM2.5 will also be performed to elucidate which source-related components are most closely associated with biological responses. This study is expected to provide the link between PM2.5 exposure and systemic inflammatory response, and will provide a cellular and molecular basis for the association of adverse cardiovascular effects with air pollution exposure.
DESCRIPTION (provide by applicant)
Estrogens, in addition to androgen, are involved in the development, growth regulation and pathobiology of the prostate. Bisphenol A (BPA), a mimic of estrogen, is now used in the manufacture of polycarbonate plastics and epoxy resins contained in a variety of consumer products. Its estrogenic effects suggest it can reprogram developing human and animal tissues, specifically those sensitive to estrogens, and alter disease risk in later life. The investigators' previous studies demonstrated that a set of genes showed aberrant methylation status in rat prostates neonatally exposed to environmentally relevant, low dose of BPA or estradiol-17-beta (E2) when compared to oil-treated controls. Among them, they found that the promoter region of phosphodiesterase 4 variant 4 (PDE4D4), encoding a cAMP-degrading enzyme, was demethylated in the prostate of adult rats as a result of neonatal BPA/E2-exposure. More recently, the investigators found that neonatal BPA/E2 treatment exerted an opposite effect on the Hippocalcin-like protein 1 (HPCAL1) in the adult prostate and caused promoter hyper-methylation of this gene. Parallel studies of PDE4D4 and HPCAL1 will generate insightful data on how intracellular cAMP contributes to prostate carcinogenesis and thereby improve our understanding of the effects of early life exposure to BPA/E2 on prostate cancer risk. The investigators propose to study PDE4D4 promoter hypomethylation together with HPCAL1 promoter hypermethylation under BPA/E2 influences as a model system to ascertain the mechanisms underpinning BPA- or E2-induced epigenetic changes in the prostate genome. The investigators hypothesize that exposure of normal prostatic epithelial cells, to BPA or E2 induces long-lasting regulation changes in specific genes epigenetically by remodeling the chromatin state and altering promoter DNA methylation patterns, thus setting the stage for neoplastic transformation. The primary focus of this application will be on epigenetic changes occurring in PDE4D4 and HPCAL1 and their association with cAMP-regulated downstream effectors and early neoplastic transformation. Results from these studies will improve our understanding of how estrogens or environmental estrogens epigenetically modify the genome, leading to development of prostate disease and/or cancer. Establishment of specific estrogen-related "epigenetically reprogramming" mechanisms will facilitate future prevention of harmful effects of related endocrine disrupters on human health.
DESCRIPTION (provided by applicant)
Breast milk is the ideal infant nutrition source. Extensive research documents the innumerable positive health outcomes and economic benefits of breastfeeding for both mothers and infants. Despite this, women in the United States do not breastfeed for as long as national recommendations. Breastfeeding is a complex process, requiring the successful integration of demographic, psychosocial, and biomedical factors. In the past, environmental chemicals with estrogenic properties have been associated with shortened durations of lactation. This potential barrier to breastfeeding, however, has not been re-examined recently or studied in detail with more than one estrogenic chemical. Although concentrations of environmental chemicals in breast milk may be low, cumulative exposure may be associated with negative lactation effects. The long range goal of this project is to examine the potential for environmental chemical contamination of breast milk to contribute to shortened lactation duration. The objectives of this proposal are to 1) determine potential factors that may impact concentrations of estrogenic environmental chemicals in breast milk, 2) characterize the changes in chemical concentration of estrogenic chemicals in breast milk over the course of lactation, and 3) establish if cumulative concentrations of these chemicals in breast milk are associated with shortened lactation duration. If environmental chemicals are shown to negatively impact breastfeeding duration, the discovery of modifiable factors to reduce breast milk contamination can be pursued. The candidate for this Career Development Award is Sheela Geraghty, MD, MS. Dr. Geraghty is a pediatrician with training in epidemiology, environmental health, and lactation. Dr. Geraghty's overall career goal is to incorporate her skills as a researcher and clinician into a program that will help women increase breastfeeding success and breastfeed longer. The Cincinnati Children's Hospital Medical Center, where Dr. Geraghty is a junior faculty member, has distinctive resources in which to help her successfully achieve this goal, including the Cincinnati Children's Environmental Health Center and the Cincinnati Children's Research Human Milk Bank. The mentors identified in this proposal are experts in the fields of lactation, environmental health, nutrition, physiology, statistical analysis, and clinical breastfeeding management. Identifying barriers to breastfeeding is in the interest of all Americans to improve child and maternal health outcomes.
DESCRIPTION (provided by applicant) This training program is entering its fifteenth year and has a full complement of 10 predoctoral trainees and four postdoctoral trainees. It achieved this size as the consequence of a merger between the current training grant in environmental carcinogenesis and mutagenesis and a separate NIEHS-funded training grant in toxicology. With the addition of appropriate new faculty, the number of preceptors is now 19. The majority of the toxicology training faculty are preceptors on this current program and have participated in mentoring predoctoral and postdoctoral trainees. The merger of the two programs has been seamless, and both trainees and preceptors have benefited from the broader scope of the program as a consequence of the inevitable cross fertilization. The major emphasis of the program remains the impact of environmental exposure on the genesis of disease, particularly cancers and also pulmonary dysfunction using molecular, genetic and toxicological approaches. The participating faculty are from four separate but interactive departments. They include the Department of Environmental Health, the Department of Cell Biology, Neurobiology and Anatomy, the Department of Molecular Genetics, Biochemistry and Microbiology, and the Department of Pediatrics, Institute for Developmental Research (IDR). Nationally, the Program fulfills an essential need to train individuals at the predoctoral and postdoctoral levels in the issues relating to biological, oncological toxicological consequences of environmental exposure. Institutionally, the Program has brought the research efforts of several laboratories into a common focus and facilitated collaborative efforts between these laboratories. Thus, the Program encourages the trainee to engage in research efforts that combine the expertise of several laboratories. This interdisciplinary approach provides the trainee with a broader-based background than is otherwise available. In the Institution, the Program has become recognized as key component in institutional efforts to support environmental health as one of its target focus areas, and the Program has been credited for spawning a much needed course in the "Biology of Cancer". Entering predoctoral trainees all have the equivalent of an undergraduate major in a chemical, biological or physical science with superior academic achievements. Postdoctoral candidates have proven academic accomplishments and hold the degrees of Ph.D., D.V.M. or M.D. Both predoctoral and postdoctoral trainees are selected from a national pool of applicants. The renewal application requests the number of predoctoral and postdoctoral trainees (10 and 4, respectively) currently supported by this training program remain the same for the next 5-year budget period. This programmatic size has proven optimal for the number of preceptors, the number of departments involved, and the extensive resources available to the trainees. It has fostered a collaborative environment between both trainees and their mentors.
DESCRIPTION (provided by applicant)
The goal of this doctoral and postdoctoral training program is to educate scientists who will provide fundamental insights into the causes and prevention of birth defects and functional abnormalities of prenatal origin. Predoctorates are selected from among interested Molecular and Developmental Biology (MDB) Program graduate students who all take classes in developmental biology, molecular genetics, cell biology and biochemistry as their core curriculum; teratology students take these classes and courses in teratology, biostatistics (new requirement), and toxicology (optional). Neuroteratology students take the aforementioned courses and courses in neuroscience. Postdoctorates also take the teratology course and all pre- and postdoctorates take an ethics in research course. Postdoctorates enter the training program through the Cincinnati Children's Research Foundation (CCRF) Office of Postdoctoral Affairs (new). All of the training faculty operate active, extramurally funded laboratories and all have experience training graduate students and postdoctorates. As a result, trainees show high research productivity, attend national meetings, and secure good positions upon completion. A high percentage of past trainees remain in science, most in teratology, thereby helping to fill the national need for scientists in this specialty. Accordingly, four predoctoral and three postdoctoral positions are requested (as before). During the current funding period, the program had its first URM trainee; in addition, several curriculum changes have been made that strengthen training in developmental genetics, and a new research building and new core facilities are being built that will add to the current research environment. Faculty membership continues to evolve with new faculty added during this period and faculty research shifting toward more investigations involving environmental agents and their effects on development. This program fills a unique niche as the only NIEHS training grant dedicated to training in teratology. This is important because teratology is not a field with its own academic home because it spans disciplines ranging from developmental biology to genetics, and toxicology to neurobiology. This renewal represents a continuation of the strengths of the program from the past and adds new initiatives that will move teratology training to the next level in understanding the mechanisms of abnormal development.
BACKGROUND
This NIEHS training program was established in 1977. Since the last competing renewal several mentors resigned from the program due to retirement, or because they are no longer accepting trainees or they are shifting focus away from areas directly relevant to the NIEHS mission. Conversely, the training program added five new faculty members (Drs. Campbell, Karp, Kuan, Wells, and Williams) who have research programs relevant to the training program. The training program has also retained seven continuing faculty members (Drs. Daston, Ma, Nebert, Potter, Scott, Vorhees, and Yutzey) from the previous funding cycle. Several curriculum changes have been made that have strengthen the training in developmental genetics, and a new research building and new Core facilities are being built that will add to the current research environment. This application represents a continuation of the strengths of the program from the past and adds new initiatives that will move teratology training to the next level in understanding the mechanisms of abnormal development
DESCRIPTION (provide by applicant)
The Molecular Epidemiology in Children's Environmental Health (MECEH) training program began July, 2001 and is in its fourth training year. MECEH is defined as the use of biological, molecular and statistical measures in epidemiological research to determine how environmental exposures impact children's health at the physiologic, cellular, and molecular levels. As the offspring of epidemiology, medicine, statistical genetics, molecular biology, and molecular genetics, molecular epidemiology serves as an umbrella for focused research in genetic and biomarker epidemiology. MECEH has 3 participating departments: Environmental Health, Pediatric Medicine and Molecular Genetics. MECEH has had continuous full enrollment with 17 trained, 6 pre-doctoral, 8 M.D./MS fellows and 3 Ph.D./M.D. postdoctoral trainees. Seven have finished or are finishing by summer 2005 and have made great professional strides with national presentation, numerous publications, grant submissions and obtaining academic research positions. This application requests support for 3 additional trainees for the 5-years from 2006 to 2011, 1 pre-doctoral and 2 postdoctoral positions.
The MECEH's long term objective is to increase the number of cross-trained epidemiologists, physician epidemiologists and molecular biologists who investigate high impact issues related to environmental exposures and complex childhood diseases. The overarching rationale for this training program has been stated in such federal initiatives as the Children's Health Act of 2000 (H.R.4365) which stressed "investment in tomorrow's pediatric researchers" (sec 1002). The MECEH has 3 primary goals: 1) provide a strong grounding in epidemiologic, statistical and wet and dry laboratory molecular methods, 2) prepare students for interdisciplinary research training and "enhance clinical research workforce training" as stated in the NIH roadmap, and 3) expand the trainee's expertise in statistical genomics, a rapidly developing area and a newer direction for this program.
The public health significance of the MECEH program is directly correlated to the increasing national awareness of the rising number of environmentally related disease such as diabetes, obesity, asthma, neurodevelopmental disorders, and prematurity among others. Thus, the public health relevance of the program is great and is directly related to the need for researchers knowledgeable across disciplines in cutting-edge methodologies in the area of pediatric environmental health.
DESCRIPTION (provided by applicant)
The goal of the Gene-Environment Interactions Training Program (GEITP) is to train pre-doctoral and post-doctoral students who will be versed in ways that both environment exposure and genetics diversity interact to alter the onset of disease. Achieving this objective requires an interdisciplinary team approach integrating an understanding of genetic diversity, epigenetic alterations, high-throughput genomics, biostatistics, biomarkers, and exposure assessment approaches. The collaborative efforts of research faculty, clinicians, postdoctoral and pre-doctoral trainees are needed in order to meet this objective. A mentorship team approach, combining the expertise of well-regarded scientists in the areas of exposure assessment, genetic variability, biomarkers of disease, and individual/tailored medicine will be used to educate trainees in multiple areas of gene-environment interactions. Pre-doctoral training will include required coursework in addition to the student's matriculated Ph.D. program, laboratory rotations with the team of mentors, and hands-on work in several areas of exposure assessment, high-throughput genetic variability measures, and biomarkers or exposure and/or disease. Postdoctoral training will include programs in laboratory and personnel management, pilot grant applications, and an intensive year long grant writing workshop to prepare them for independent research. All trainees will be required to attend "Technologies in genomics, exposure, a biomarkers" workshop, which will be created as part of this program, and present research results at an annual GEITP Student Symposium. This program will include a dedicated governance structure to assure that appropriate trainees are recruited, education goals are met, and the aims of this grant are achieved.
DESCRIPTION (provided by applicant)
The ICWU Center coordinates the delivery of high quality, cost-effective training by 38 worker-trainers, using sound principles of adult education and yearly developing new curricula to meet the site requirements under Integrated Safety Management (ISM) at Hanford, Oak Ridge, Kansas City MO, Amarillo TX, Fernald and Albuquerque. The long-term organizational goal is to continue the institutionalization of the program within member unions, Councils and contractor management at DOE sites, as has been accomplished at Hanford HAMMER, Oak Ridge and Kansas City. As an immediate educational goal, DOE worker-trainers will continue delivering HAZMAT training to thousands of DOE workers who are daily exposed to a wide variety of hazardous substances, meeting ISM requirements.
The long-term educational goal is to provide all students with the confidence, relevant tools and problem solving skills to identify inadequacies in their facilities' hazardous materials programs.
ICWU proposes to yearly conduct 230 classes for 3,351 trainees for 32,840 contact hours (1,150 classes for 16,755 trainees for 164,200 contact-hours over the 5-year period), including trainer development classes. These are 66 One-day Site Refresher classes, 3 One-day Chemical Emergency Response classes, 7 40-hour Hazardous Waste Operations classes, 11 RCRA 24-hour classes, 6 Bridge classes, 120 Respirator classes, Trainer Development classes, Consortium DOE Trainers Exchange, and continue minority community outreach. Long-term evaluation includes the continuance of an evaluation project which demonstrates participants' ability to make workplace changes. With regards to Quality Control, quality curriculum will be ensured through compliance with NIEHS, DOE orders and OSHA requirements. Joint Labor-Management goals are to continue to work with, train and market these programs to site management.
Plans for new projects include the following: Annual Multi Awardee DOE Trainers Meeting; Chemical Release Software training; Disaster Site Worker; and train 28 DOE trainers in the 5600 Disaster Site Worker and the 500/510 Construction Worker Train-the-Trainer programs (1,120 contact hours) in order to establish 5 Disaster Site Worker Training Centers. The applicants intend to provide professional level education to active site trainers.
DESCRIPTION (provided by applicant)
The ICWU is applying for 2 programs: HWWT ($2,594,449 Yr1; $13,765,662 Total) and HDPT ($606,253 Yr1; $3,218,678 Total). These programs will reach 2,466 trainees in 145 classes for 55,520 contact hours. This would result in 12,330 trainees in 725 classes for 277,600 contact hours over the 5-year period of the grant. Over the last 17 years, the ICWU Center, based in Cincinnati OH, and its nationwide union Consortium has built a nationally recognized HAZMAT training program specializing in chemical emergency response programs to protect collateral duty emergency responders. The Consortium members are: ICWU, UFCW, Coalition of Black Trade Unionists (CBTU), American Federation of Teachers (AFT), American Nurses Association (ANA), GCOHC, American Federation of Government Employees, UC and IAM. The long-term organizational goal is to institutionalize a program within member unions and build employer support. The immediate educational goal is to deliver emergency response training to industrial, educational, government and health care workers with collateral duty to respond to chemical emergencies. The long-term educational goal is to provide students with the confidence and problem-solving skills to identify inadequacies in facility chemical emergency response programs. The applicant will evaluate the effectiveness of workplace improvements through pre and post training evaluation. They will expand their programs to protect school and hospital employees who respond to spills within their facilities and protect health care workers from contaminated patients after catastrophic unintentional incidents. Hospital "First Receivers" are recognized by OSHA as covered by the HAZWOPER standard and in need of training. The ICWU consortium will continue the application of blended learning and expand their already substantial multi-grantee efforts into new areas. HWWTP specific aims are to, continue to develop worker-trainers, using Adult Education Techniques; expand Minority Outreach with CBTU and Latino workers; collaborate with other grantees; train industrial workers, government workers, school personnel, non-WMD (weapons of mass destruction) First Receivers; and expand into Spanish language Hazmat training, Chemical Release Software, mold, Worker and Community Outreach and Workplace Shelter in Place. The HDPTP aims include, continuing WMD First Receivers training and expanding to Disaster Site Worker training, Worker and Community Outreach and Workplace WMD Shelter in Place and the use of Chemical Release Software.
Hazardous Waste Worker Training Program (HWWTP)
DESCRIPTION (provided by applicant)
The Midwest Consortium for Hazardous Waste Worker Training will conduct HWWT and HDPTP programs. The University of Cincinnati requests for HWWT $11,021,001 (total, direct + F&A) for the project period. The University of Cincinnati requests for HDPTP $2,100,109 (total, direct + F&A) for the project period.
In the HWWT program, HAZWOPER training will be provided in eight states of the Midwest and on the Reservation of The Three Affiliated Tribes. We will continue to maintain and update 19 programs while adapting them to new challenges such as methamphetimine lab cleanup and enhancing them to interactive tabletop exercises and e-tools. Change in knowledge and skills and the impact of training will be documented. We will provide 4,919 programs to 94,933 trainees for a total of 673,472 contact hours.
In the HDPTP initiative, we will immediately deliver CERT training, Emergency Operations Center Programs, Hospital Decon programs, introduce a Bioterrorism tabletop and provide on-line training in Crime Scene Integrity and Reporting Environmental Releases. A partnership with the Amalgamated Transit Union (ATU) is initiated to train their members nationwide through a Train-the Trainer program. 30 trainers and 750 participants will be trained in the first year during the direct delivery of the HDPTP initiative. Additional participants will be trained on-line. At the end of the first year, we will have completed the work to multiply the training delivery capabilities of the Midwest Consortium and the ATU for Years 02-05.
Hazardous Waste Worker Training Program (HWWTP)
DESCRIPTION (provided by applicant): Marietta-Parkersburg Metropolitan Area is a rural Appalachian community. The area has been home to the only manganese (Mn) refinery in the US and Canada for over 50 years. The residents are very concerned about the quality of their air and related health effects. Caroline Beidler, a local resident, has created and directs, Neighbors for Clean Air (NCA). Beidler has sought the assistance of Dr. Erin Haynes at the University of Cincinnati to help her and her community understand the health effects of chronic exposure to air Mn. Together we have conducted a community-wide survey to evaluate the community's level of concern about environmental air pollution in their region caused by industrial emissions and to learn about their perceptions of air quality and its impact on their health. Their sources of information related to local industrial emissions, their level of trust for these information sources, and how they rate the level of knowledge from each information source was also evaluated. We found that the community relies upon local media for air quality information, yet these reporters are not trained in science or environmental journalism. The community also uses websites to search for information on industrial emissions and health effects; however, reliable sites are difficult to find, navigate, and interpret. Thus, Beidler and Haynes have created a "University of Cincinnati-Marietta- Parkersburg Metropolitan Area Reciprocal Research Relationship" Project. The goal of this project is to create reciprocal research partnerships to determine the health effects associated with the Marietta-Parkersburg Metropolitan Area community's exposure to Mn while engaging the community in the research process to ultimately inform regional and national policy for Mn emissions. Our main objective is to implement and evaluate a multifaceted Community Based Participatory Research (CBPR) program that bridges the gap between an affected community and the research scientists. The following specific aims for the project are: Specific Aim 1: Create reciprocal training and research opportunities for the Marietta-Parkersburg Metropolitan Area community and University of Cincinnati scientific researchers; Specific Aim 2: Create a reciprocal educational relationship among Marietta-Parkersburg Metropolitan Area journalists (radio, newspaper and TV reporters), University of Cincinnati research scientists and environmental journalists to establish a well-defined network between journalists and research scientists; and Specific Aim 3: Evaluate the success of the overall community-research partnership through qualitative research methods. Given the community's intense interest in understanding their air quality and resulting health effects, the developing partnership between UC and the Marietta-Parkersburg Metropolitan area, and the opportunity for community involvement in innovative research studies, we anticipate that the results of this proposal will provide a model community-research partnership with reproducible deliverables for national and international community-based participatory research studies.
DESCRIPTION (provided by applicant): Manganese (Mn) is an essential element, yet is neurotoxic in excess. Despite the fact that infants and young children may be at greater risk for Mn neurotoxicity than adults, very few studies have evaluated the effect of chronic Mn exposure on child development. Marietta, Ohio, a rural Appalachian community, has been home to the only metal refinery in the US and Canada for over 50 years. In response, the community formed Neighbors for Clean Air (NCA). The University of Cincinnati (UC) has been asked by NCA to assist them in understanding their exposure and related health effects, if any. For this project, Marietta Community Actively Researching Exposure Study (CARES), the community has defined the research agenda, determining the research questions, objectives, specific aims and identifying resources available within the community to conduct the research. Members of the community will be engaged in all facets of the research project, including environmental sampling, neurobehavioral assessment, project coordination, and collection of biological specimens. The primary objectives of this application are to examine biological indicators of Mn exposure in children and understand the effects of chronic manganese exposure in children. UC will provide training, oversight and expertise in environmental health research, to address the following hypothesis: Exposure to Mn during early childhood is significantly associated with neurodevelopmental deficits in high exposed children ages 7-8 years compared to an age matched low/unexposed group. In order to test the hypothesis, the following community-driven specific aims will be accomplished. Specific Aim 1: Assess environmental Mn exposure within a high Mn exposure community, Marietta, OH and a low Mn exposure community, Cambridge, Ohio. Specific Aim 2: Evaluate comparisons of the neurobehavioral effects in children exposed to Mn in a high Mn exposure community, Marietta, OH and a low Mn exposure community, Cambridge, Ohio. Specific Aim 3: Evaluate comparisons of the neuromotor effects in children exposed to Mn exposure community, Marietta, OH and a low Mn exposure community, Cambridge, Ohio. Given the unique susceptibility of children to toxicant exposure and the opportunity to study Mn exposure in a chronically exposed community, we anticipate this project to significantly impact local and national public health policy related to Mn exposure. Project Narrative Exposure: to manganese (Mn) can result in neurological deficiencies, and here is great potential for global exposure to Mn in the form of MMT, an anti-knock agent to replace lead in gasoline. The Marietta Community Actively Researching Exposure Study (CARES) is a community-based study designed to explore the health effects of long term Mn exposure in children. PUBLIC HEALTH RELEVANCE: Exposure to manganese (Mn) can result in neurological deficiencies, and here is great potential for global exposure to Mn in the form of MMT, an anti-knock agent to replace lead in gasoline. The Marietta Community Actively Researching Exposure Study (CARES) is a community-based study designed to explore the health effects of long term Mn exposure in children.
DESCRIPTION (provided by applicant): The incidence of obesity has risen dramatically over the last few decades. In addition to high caloric food intake and sedentary life style, the role of environmental factors is gaining credence. Bisphenol A (BPA) is a monomer of polycarbonate plastics used in many consumer products. BPA exerts multiple estrogenic-like actions and is detectable at H 1-20 nM in serum from the majority of tested individuals worldwide. We found that BPA at low nM doses suppresses adiponectin release from human adipose explants, with visceral explants from morbidly obese patients showing the highest sensitivity to the suppressive effect of BPA. Adiponectin is an adipocyte-specific hormone which plays a key role in metabolic homeostasis. Lower serum adiponectin levels are associated with the manifestation of the metabolic syndrome. Thus, any factor which suppresses adiponectin should subject the exposed individuals to increased risks of developing diabetes or cardiovascular disease. To support the premise that endocrine disruptors increase the risk of obesity-associated diseases, studies with human adipose tissue are critically needed. Our first objective is to establish that BPA, at environmentally relevant doses, inhibits adiponectin production and release from human Adipose tissue. Our second objective is to explore whether BPA rapidly inhibits adiponectin release and suppresses adiponectin expression by interacting with classical and non-classical estrogen receptors. Specific aim 1 will compare the suppressive effects of BPA and estradiol on adiponectin gene expression and release from visceral and subcutaneous adipose explants from obese and non-obese patients. Specific aim 2 will examine the mechanisms underlying acute and chronic effects of BPA, focusing on the roles of estrogen receptors (ER1 and/or ER2), G-protein-coupled receptor 30 (GPR30) and peroxisome proliferator- activated receptor 3 (PPAR3). The second aim will be accomplished using primary human adipocytes or LS14, our novel human adipocyte cell line. Our long term goal is to establish the metabolic effects of BPA in human adipocytes and generate solid evidence that can be used by regulatory agencies to determine whether BPA in the environment is hazardous to human health. PUBLIC HEALTH RELEVANCE: This study examines the effects of bisphenol A, an endocrine disruptor that is common in the environment, on the production and release of adiponectin from human adipose tissue and adipocytes. Adiponectin is an adipose-specific cytokine that plays a critical role in metabolic homeostasis and its suppression is associated with increased risk of developing diabetes and cardiovascular diseases that are associated with the metabolic syndrome.
DESCRIPTION (provided by applicant)
The goal is to use metabolomics coupled to mass isotopomer analysis to study the metabolic and oxidative stress exerted by two related compounds, i.e.,1,4butanediol (14BD) and gamma-butyrolactone. These industrial solvents are used extensively in the chemical industry in building materials and in consumer products. Lastly, they are precursors of the dangerous drug of abuse gamma-hydroxybutyrate (GHB) which is very popular among young people. The investigators have outlined a strategy to accelerate the disposal of gamma-hydroxybutyrate in the liver. The coupling of metabolomics to mass isotopomer analysis will provide new avenues for understanding xenobiotic stress. The aims are:
1. To determine if compounds identified by the metabolomics approach provide new insight into the metabolism of 14BD and GHB. The investigators will generate a database of metabolic information obtained by mass spectrometric analyses of plasma, urine and liver of control and 14BD- or GHB- exposed rats. Multivariate statistical methods will reduce the dimensions of the data set of unknown peaks, detecting those that discriminate between treatments. A number of techniques will be evaluated, including Principal Component Analysis, Fisher Discriminant Analysis and Partial Least Squares.
2. To study the temporal patterns of concentration and mass isotopomer distribution of metabolites extracted from (i) isolated rat livers perfused with unlabeled and uniformly 13C-labeled 14BD and GHB and (ii) the plasma, urine, liver and kidney of rats infused with these compounds. The rats will be normal or pre-treated with compounds that interfere with the metabolism of 14BD and/or GHB (ethanol, methylpyrazole). The patterns of change in the profiles following an intervention will be analyzed with a new software tool, Metran, which produces flux estimates with statistical confidence from a pathway model and isotopomer data. The data will also be analyzed by multivariate statistics described in aim 1 to identify discriminating isotopomers, which may provide clues to regulatory mechanisms.
3. To test the hypotheses that the metabolism of GHB in liver and kidney can be accelerated by glucuronolactone, precursors of alpha-ketoglutarate or by taurine. This will involve an investigation of the mechanisms of the enzymes that catalyze the conversion of GHB to succinic semialdehyde.
4. To characterize the response of the liver to oxidative stress by a noninvasive technique to measure the turnover of glutathione in liver. This will be achieved by administering low doses of 2H20 and acetaminophen, followed by measuring the 2H-labeling of urinary acetaminophen-glutathione adduct.
DESCRIPTION (provided by applicant):
Environmental exposure to endocrine-disrupting agents, or xenoestrogens, can increase the risk of developing breast cancer. Animal and population studies suggest an imprinting phenomenon whereby early exposure of xenoestrogen can lead to tumorigenesis later in life. The molecular mechanism by which these environmental stressors can transform breast genomes is not well understood. Our preliminary data prompt us to hypothesize that epigenetic alteration, in the form of CpG island hypermethylation, transmits this imprinted information to the progeny of undifferentiated cells pre-exposed to xenoestrogens. Specifically, we propose that immature cells located in the stem/progenitor compartment of the human breast are prime targets of this environmental insult. In Specific Aim 1, primary breast stem/progenitor cells will be exposed to xenoestrogens - diethylstilbestrol, bisphenol A, or 17(3-estradiol in an in vitro system. 'Global analysis is expected to identify altered methylation status in 1-2% of ~29,000 CpG islands analyzed. These epigenetic events can be the direct results of exposing stem/progenitor cells to xenoestrogens. The epigenetic memory of this injury is then transmitted to differentiated epithelial cells and in turn leads to breast tumorigenesis in a xenograft model. In Specific Aim 2, we will functionally determine whether the prolonged exposure of these endocrine chemicals to stem/progenitor cells disrupt the homeostasis of estrogen signaling and triggers an epigenetic cascade in its downstream targets. Polycomb repressors can be recruited to promoter CpG islands followed by the addition of DNA methyltransferases at these promoters. Acquired DMA methylation, as a result of increased local methyltransferase activities, marks the heritable gene silencing. In Specific Aim 3, we will demonstrate that CpG island hypermethylation induced by xenoestrogen exposure is also observed in clinical samples. The presence of these molecular alterations in primary breast tumors may constitute a xenoestrogen epigenotype(s). In this regard, patients exhibiting this epigenotype are likely exposed to xenoestrogens in their early lives. In addition, low levels of these methylation changes may exist in normal looking mammary epithelial, leaving a field of cancerization in the human breast. This type of CpG island hypermethylation can be tracked as molecular relics using a mathematical modeling approach developed in our laboratory. We will develop the model further to recreate the history of xenoestrogen- induced breast tumorigenesis, from pre-neoplastic lesions to hyperplasia to carcinoma in situ to invasive carcinoma. Clinical sensitivity and specificity of potential CpG island loci pinpointing the xenoestrogen epigenotype will be provided as a quantitative milestone for this U01 project. These loci are future biomarkers for early breast cancer detection and are putative biosensors to environmental estrogens.
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