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Record Count: 20
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DESCRIPTION (provided by applicant): Chronic exposure to Cd2+ in humans causes kidney failure that is characterized as a Fanconi syndrome, in which an array of Na+-dependent nutrient transport processes is inhibited. Cd2+ causes a sub-lethal, concentration-dependent reduction of Na+-glucose co-transport in mouse kidney cortical cells that is correlated with a suppression of SGLT1 and 2 mRNA transcription. Results from a variety of sources convincingly argues that phosphorylation signaling links Cd2+ with targets in the SGLT system to inhibit Na+- glucose co-transport. We hypothesize that Cd2+ down-regulates sgltl and 2 gene activity at least in part through the activation of protein kinase C. Direct interaction of Cd2+ with protein kinase C in turn, initiates a signaling cascade that culminates in the phosphorylation and down-regulation of Sp1 and/or SGLT1 and 2 proteins. The signaling pathway is hypothesized to involve mitogen activated kinase (MARK) cascades. Further, we hypothesize that Cd2+ directly displaces Zn2+ from Sp1 and, thereby, contributes to the Cd2+- dependent inhibition of SGLT1 mRNA synthesis. Our overall objective is to understand the cellular mechanism of Cd2+-inhibition of SGLT activity beginning at the molecular biological level and concluding with the definition of the molecular site(s) of binding and action of Cd2+. In the process, basic features of kidney sgltl transcriptional regulation will be addressed. Our Specific Aims are 1. To ascertain whether the Sp1 binding sites in the sgltl promoter are the critical sites that respond to cellular exposure to Cd2+. 3. To identify changes in phosphorylation state of transcription factor Sp1 as one of the immediate molecular events that links cellular Cd2+ ion to its inhibitory effects on sgltl transcription. 4. To determine the roles of PKC and MAP kinases in the signaling pathway between Cd2+ and SGLT1 mRNA synthesis. 5. To define relevant chemical properties of cellular Cd2+-binding proteins implicated in the cellular mechanism of control of sgltl transcription by Cd2+, particularly Sp1, PKC, and metallothionein. 6. To obtain 3-dimensional structural information about Cd2+-binding domains of Sp1 and protein kinase C. The proposed studies combine molecular biological, chemical, and structural experiments in order to achieve an integrated bio- chemical understanding of the mechanism of Cd2+ inhibition of Na+-glucose co-transport in the kidney.
DESCRIPTION (provided by applicant): Understanding the molecular details of
dioxin/Ah receptor (AHR) signal transduction will yield a number of benefits.
First, identifying modifiers of AHR signaling will help explain the tissue and
species specificity of dioxin toxicity and thus will aid attempts to predict
the human and environmental risk that results from exposure to these
pollutants. Second, the AHR is a prototype of a large super family of
environmental sensors. What we learn here will shed significant light on a
variety of environment-gene interactions. Third, it is likely that the AHR will
be involved in a variety of human disorders. Detailed information about
signaling can guide the pharmacology necessary to develop related therapeutics.
Finally, identifying endogenous activators of the AHR will provide one of the
most significant clues as to how and why this protein signals in development
and begin to explain the conservation of this protein throughout evolution. In
an effort to answer these questions, we propose the following specific aims:
Aim 1: Identify/understand modifiers of AHR signaling through the use of
genetic screens in the yeast, S cerevisiae. We will employ two screening
approaches in yeast. First we will use a library of deletions to determine the
role of each yeast gene product on AHR signal transduction. Second, we will
expand upon these screens by using mammalian cDNAs to perform high copy
modifier screens in yeast. Identified modifiers will then be characterized
using both statistical clustering of their pharmacological action, as well as
biochemical approaches to elucidate mechanism. Aim 2: Understand the mechanism
of AHR modifiers through the use of signaling kinetics in mammalian cell
culture. We propose to use fluorescently tagged molecules and reporters to
elucidate the cellular mechanisms of AHR modifiers using a mammalian culture
system. Once developed, this system will also serve as the basis for an
activator screen described in Aim 3. Aim 3: Determine how the AHR signals
during development. The phenotype of Ah null mice suggests the existence of a
developmental activator of the AHR. To identify this factor, we propose to: 1)
identify those developmental sites where the activator is most likely to be
expressed; 2) screen for cDNAs encoding protein factors that activate the AHR
using an activator trap protocol in cell culture; and 3) characterize the
soluble factor derived from murine heart that activates the AHR in cell
culture. Aim 4: Complete the high-resolution domain mapping studies of the AHR.
DESCRIPTION (provided by applicant): Thousands of recreational beaches along United States coasts have experienced water quality advisories or closures due to elevated levels of fecal indicator bacteria, such as Escherichia coli (E. coli). However, this methodology may not reflect the number of actual pathogens present or the potential human health risk. Also, little is known about the survival of biological contaminants in the beach sand, which may impact monitoring results. In order to validate use of E. coli as an indicator organism, it is important to understand the physiological mechanisms for adaptation and survival. This proposal encompasses three major aims. Aim 1 focuses on continuation of a 4-year study to evaluate the E. coli burden at a heavily trafficked urban beach on Lake Michigan. Bacterial counts will be determined throughout the year. Clonal typing will be performed to determine the extent to which selected populations from contamination sources can adapt to and colonize the beach sand habitat. Aim 2 investigates the induction and modulation of two physiological responses that may confer protection against desiccation stress in sand; namely trehalose synthesis and shifts in porin protein expression. Sand model plots will be loaded with a beach sand E. coli isolate and placed on an environmental green roof for exposure to the same conditions as nearby beachfronts. Survival outcomes for E. coli will be determined under fluctuating desiccation/rehydration conditions and in the presence or absence of resident microbiota. The gene products for porin protein ompC and the end-product of the ots operon (trehalose) will be measured in sand eluant samples taken throughout the study. Aim 3 focuses on long-term survivorship of selected E. coli from the beach site and for isolates loaded in short term (<7 days) and long-term (>14 days) sand plot models. Expression of fitness advantage traits (e.g. osmotic tolerance) will be evaluated under environmental and controlled growth conditions of varying temperature and desiccation and in competition assays with known laboratory strains. Throughout all aims we will examine modulation of genes associated with growth phase transitions (e.g. rpoS, dps, katE). Findings from these studies will provide public health officials with more contextual information in which to interpret water monitoring data, and will allow beach managers to formulate new strategies for abatement of coliform pollution in recreational waterfronts.
The cellular mechanisms involved in the survival of the fecal indicator organism E. coli in beach sand, a habitat of increasing interest, are relatively unknown. Almost all designated public beaches in coastal regions will have monitoring programs for fecal pollution implemented by 2007. Understanding the environmental reservoirs and determinants of E. coli die-off will be important for interpreting monitoring results of beaches and discerning whether elevated E. coli levels reflect human health risk.
The Marine and Freshwater Biomedical Sciences Center of the University of Wisconsin- Milwaukee is an extramural Center of the NIEHS, The theme that unifies Center activities for this renewal proposal remains the development and exploitation of aquatic biomedical model systems for the study of significant human environmental health problems. In order to advance this theme, the overall Center objectives include, (1) providing a multifaceted array of support for investigators that enhances innovative, multidisciplinary, and interactive research with aquatic organisms on significant problems related to human environmental health, (2) compacting scientists and their research with the public in order to enrich the community' s understanding of environmental health and its importance. Among key specific aims of the Centers cores are the following: Administration (a) To assemble a first rate group of Center investigators from a variety of complementary disciplines who interact and collaborate with one another on important research problems in the area of environmental health. (b) to provide Center investigators with a stimulating environment that fosters innovative, cooperative research and communication. (c) To continue to find additional sources of funding to support the development of the Center's program. Facility and Service Cores (a) To provide increasingly sophisticated staff and facilities support to investigators in the design and implementation of experiments, (b) to expand the support services including techniques in fish research and molecular biology and microscopy which otherwise would be unavailable to researchers, and (c) to offer expert technical support for outreach and education projects. Research Cores (a) To continue to develop excellent, interacting groups in Metals/Neurobehavioral Toxicology and in Signal Transduction/Endocrine Disruption Toxicology. (b) To build the premier program in zebrafish toxicology. Community Outreach and Education Program (a) To increase science and environmental health literacy among precollege youth, (b) To offer research opportunities to minority college students in environmental health science disciplines. (c) To increase the kmowledge and involvement of minority communities in environmental health issues. (d) To increase competency of nurses and public health practitioners in environmental health practice.
Congenital heart disease is a major cause of mortality and morbidity. Exposure to halogenated hydrocarbons, specifically trichloroethylene (TRI), during pregnancy has been associated with congenital heart disease in animal models and retrospective epidemiology studies. Multiple studies support trichloracetic acid (TCA), a TRI metabolite, as the metabolite, as the more proximate teratogen. Overall hypothesis is that commonly observed levels of trichlorinated hydrocarbon exposure during human pregnancy are associated with an increase for offspring congenital heart disease and that genetic and phenotypic differences in the key enzymes responsible for the formation of TCA are associated with differences in offspring susceptibility. The specific aims are to 1) prospectively determine the risk of congenital heart disease from commonly observed levels of maternal exposure to trichlorinated hydrocarbons during pregnancy and 2) determine the differences in the offspring risk for congenital heart disease from maternal intersubject variation in the enzymes catalyzing the disposition of TRI among mother-infant pairs with documented trichlorinated hydrocarbon exposure during pregnancy. To complete these aims, we will determine the presence of congenital heart disease by performing echocardiography on infants selected using a stratified recruitment strategy based on 1) maternal urinary trihalogenated hydrocarbon concentrations measured during pregnancy using a sensitive GCMS assay 2) maternal CYP2E1 genotype for a polymorphism associated with increased CYP2E1 activity in the presence of inducers and 3) maternal ADH2 genotype, a polymorphism which impacts the metabolism of other small molecular weight hydrocarbons. In addition, because multiple environmental factors alter CYP2E1 metabolic ability, some by post-transcriptional mechanisms, we will evaluate maternal white blood cell microsomal immunoreactive CYP2E1 during pregnancy as an important biomaker of increased offspring risk. Because the genotypes of interest are more common in the African-American population and because TRI emissions are documented in the Milwaukee area, an urban Milwaukee, African-American population will be studied. Maternal exposure to volatile organic solvents and ethanol will be measured and induced as potential confounders. Ultimately, the data generated in this proposal, will leaf to a better understanding of the genetic and environmental mechanisms determining susceptibility for congenital heart disease and provide the knowledge necessary for future public health prevention/intervention strategies.
DESCRIPTION (provided by applicant): Susceptibility to common diseases such as breast cancer is complex. Minimally the etiology of susceptibility is centered on a large number of interacting genetic elements which individually and collectively interact with environmental components. In order to assign risks to individuals, in contrast to populations, it will be necessary to refine an individual's inherent risk alleles and their interaction with each other and the individual's environment. To accomplish the goals of individual risk estimation and its mitigation, disease-specific integrated genetic systems networks are needed. Work will focus on breast cancer genome-wide association studies (GWAS) SNPs in ~10% of the human genome that is homologous to the highly defined mammary susceptibility QTLs in the rat. High throughput gene expression measurements from at least 50 reduction mammoplasty human mammary epithelial cells (HMEC) samples from healthy women together with full genome SNP genotypes will be obtained. Expression quantitative trait loci (eQTL) will be identified and integrated with GWAS results for breast cancer risk. The integrated data sets will be used for three important purposes. First, they will be used to assign function to a group of tag SNP alleles from breast cancer GWAS. The second will be to establish network systems models that suggest potential causal relationships among SNPs and downstream phenotypes. The third application of these integrated data sets will be to prioritize suspected but not yet validated tag SNP risk alleles for further validation studies using Wisconsin breast cancer case-control DNA samples (n = ~7,000). Next, investigating the effects of environmental factors on gene expression in HMEC will further develop and functionally explore the groups/networks of transcripts identified above. Primary cultures of HMEC will be exposed to xenobiotics chosen using prior knowledge. The expression levels of genes of interest will be evaluated asking if such agents (toxic and preventive) can modulate the expression of important groups of transcripts associated with GWAS SNPs and if exposure significantly alters network structure. GWAS SNPs that are associated with gene expression changes caused by specific xenobiotics will be used to determine if stratification by these SNPs modifies relative risk for that environmental agent in the Wisconsin case-control population. Finally, in vivo validation studies using the congenic rat mammary carcinogenesis models initially used to focus human studies will be conducted.
PUBLIC HEALTH RELEVANCE: The goal of this project is to develop an integrated approach combining global genetic information together with environmental exposure to form a network model that begins to describe the etiology of breast cancer. Such a model, when complete, could allow us to move from the estimation of population risk for breast cancer to individual risk. This model will also provide functional information underlying genetic/environmental risk that could lead to strategies for risk reduction to this disease.
DESCRIPTION (provided by applicant): The objective of this proposal is to understand how a novel action of estrogen and man-made xenoestrogens affect the initial patterning and development of the nervous system. This will expand our knowledge of the signaling pathways that regulate a specific type of neuron. It also identifies a potential cellular target of endocrine disruptor molecules, which may be related to long-term effects on organismal development. These studies utilize Xenopus laevis, an NIH model organism. The first specific aim tests whether the amount and location of estrogen plays a role in the normal development of dopaminergic neurons and if exogenous estrogen and xenoestrogens are localized near the affected neuron population. The second aim identifies the molecular signaling pathway utilized by estrogen to affect dopaminergic neurons through loss- and gain-of-function experiments with different estrogen receptors. These experiments use antisense morpholinos to knock down specific estrogen receptors and the RNA of individual receptors to achieve their overexpression. The effects will be measured on the dopaminergic neuron population during normal development and in response to additional estrogen. The third aim tests the long- term consequences of brief embryonic exposure to estrogen and xenoestrogens. Prolonged exposure to estrogen during later periods of development affects sex determination and metamorphosis in Xenopus. Given the pronounced cellular effect on dopaminergic neurons after brief embryonic exposure, they could mediate these developmental changes. If estrogenic molecules including Bisphenol A and Octylphenol can alter sex determination and metamorphosis in developing Xenopus, it would indicate there are potential hazards associated with embryonic exposure to estrogenic compounds in a wide range of vertebrates, including humans. Relevance to public health. Endocrine disrupters are man-made molecules present in the environment that interfere with the normal hormonal signaling pathways in humans and animals alike. This proposal examines the molecular mechanisms used by estrogen and endocrine disrupters that mimic it (xenoestrogens) to affect the initial development of a specific population of neurons and its potential long term effects on the organism.
Crisp Terms/Key Words: Xenopus, central neural pathway /tract, nonmammalian vertebrate embryology, estrogen, developmental genetics, gene expression, sex determination, metamorphosis, radioimmunoassay, neurogenesis, antisense nucleic acid, oligonucleotide, dopamine, protein structure function, radiotracer, estrogen receptor, toxin, protein localization, endocrine disrupting compound
DESCRIPTION (provided by applicant): This is a proposal to understand Ah receptor (AHR) biology and also an attempt to develop new tools for the toxicologist. In this proposal, we will couple recombinant mouse models, liver cell isolation techniques and modern genomic technology to identify those transcriptional outputs that define the AHR's role in the hepatotoxic effects of potent agonists like 2,3,7,8-tetrachlorodibenzo-pdioxin ("dioxin"). In parallel, we will use the AHR as a model system to develop a new generation of tools, reagents and resources that will aid biologists in their efforts to understand toxicant action. Our specific aims are: Aim 1: Develop cDNA clone sets and microarray technologies (MAT) that are optimal for the study of AHR signaling in parenchymal and nonparenchymal cell types. Aim 2: Develop statistical tools for the optimal design of toxicogenomics studies, as well as to interpret these studies by incorporating data from mutant animal models, pathology and the biomedical literature. Aim 3: We will test the hypothesis that various cell types are interacting and that this interaction can be detected through the presentation of waves of temporally linked transcriptional change. Aim 4: Elucidate the roles of the candidate genes, AHR, Cyplal, Cypla2, Cyplbl, TNF-a and IL6. Using recombinant mouse models, each of these genes will be tested for roles in aspects of hepatotoxicity or for roles in dioxin-induced signaling across hepatic cell types. Aim 5: Test the idea that global aberrations in gene expression can serve as a new definition of toxicity. We will also test the idea that patterns of gene expression will define various pathologies. Aim 6: The MAT we develop will have significant application across toxicant classes. Therefore, we will develop a resource where toxicologists can view the transcriptional responses to dioxin and classic hepatotoxicants.
DESCRIPTION (provided by applicant): The objective of this proposal is to understand the mechanisms by which environmental chemicals influence liver tumor promotion. To accomplish this task, we propose to use the prototype carcinogen, 2,3,7,8-tetrachlorodibenzo-
p-dioxin (dioxin), as a promoter of hepatocellular carcinoma in the two-stage mouse model. The choice of dioxin is related to its biological potency and the large volume of genetic and pharmacological evidence that suggests its effects are mediated through a single ligand-activated transcription factor known as the Ah receptor (AHR). Given the central nature of this signaling protein, efforts will be made to elucidate the molecular details of the dioxin-AHR pathway as it relates to liver tumor promotion. To this end, we will first optimize the murine model of liver tumor promotion, with particular emphasis on genetics and statistical power. We will then use gene-targeting approaches to manipulate various functional domains and expression patterns of the AHR, as well as its heterodimeric partner ARNT. In particular, focus will be on determining if dioxin's tumor promoting activity is a cell autonomous process and if it is the direct result of the AHR's or ARNT's activity as transcription factors. This understanding will guide the later experiments that will be directed at identifying those particular transcriptional outputs that lie in the pathway to tumor promotion. In this last respect, emphasis will be placed on direct test for the involvement of candidate genes such as Cyp1a1, Cyp1a2 and Cyp1b1, as well as the IL1-like inflammatory cytokines. As a backup approach, we will begin to develop novel screening approaches to identify candidate genes using high throughput transcriptional profiling of preneoplastic lesions and SiRNA technologies to elucidate functional roles in promotion.
DESCRIPTION (provided by applicant): Multiple sclerosis (MS) is a chronic debilitating disease that is associated with demyelination and axonal degeneration within the central nervous system. MS affects over 450,000 people in the United States. In Wisconsin there are over 11,000 people with MS, making it a highly prevalent disease. MS is a complex disease, in which environmental factors act together in a genetically susceptible person to cause disease. The goal of this exploratory project is to test the hypothesis that conditioning on genetic risk factors will assist in identifying the environmental trigger of MS. We will test our hypothesis using case-control association studies in unique data sets. These consist of 300 MS patients and 600 unrelated controls. All participants, including patients and controls, are born in Wisconsin, and they and their families have lived in the same region for several generations. Specifically, most patients had spent the putative critical exposure period in Wisconsin and most controls are selected from a population-based cohort. This study has three aims. (1) Complete recruitment of sporadic cases with MS, and healthy controls from Wisconsin. (2) Stratify the newly enrolled participants according to the presence or absence of the HLA- DRB1*1501 allele. (3) Compare the association between MS and two environmental risk factors, childhood infections and diet, in MS subjects who do and do not carry the DRB1*1501 allele. Multiple sclerosis (MS) is the most common neurological disease affecting young people of Northern European descent. MS is a complex disease, in which environmental factors trigger the onset of symptoms in persons who are genetically vulnerable to the disease. The goal of this study is to identify environmental triggers in patients known to carry the genetic susceptibility for MS. We believe that the study of the environmental trigger in a clinically and ethnically homogeneous study sample from the same geographic region will help identify these triggers. This in turn will allow disease control and prevention.
DESCRIPTION (provided by applicant): Oxidative stress is an imbalance in which free radicals and their products exceed the capacity of antioxidant defense mechanisms. The harmful reactive compounds generated by oxidative stress are associated with neuronal cell death following acute insults and are also believed to be a principle factor in the development of many chronic neurodegenerative diseases such as Alzheimer's, Parkinson's (PD), Huntington's and Amyotrophic Lateral Sclerosis. The expression of many neuroprotective phase II detoxification enzymes and/or antioxidant genes is governed by the antioxidant responsive element (ARE). ARE-dependent gene expression is induced by the transcriptional factor, Nrf2, and is considered to be a novel and important pathway that confers protection to a variety of oxidative stress-related neurodegenerative insults. The long- range objective of the laboratory is to evaluate the regulation and cell-specific expression of ARE-driven genes and the potential role of these genes in prevention of neurodegeneration. In order to develop potential therapeutic strategies targeting Parkinson's disease through activation of the ARE, small molecules that penetrate the blood-brain barrier and robustly activate ARE will be required. We will use primary cortical cultures from Nrf2 knockout and wild-type mice as an in vitro system to examine potential chemical activators of the ARE. We will test whether compounds identified as potent ARE activators can attenuate nigrostriatal lesions in both 6-hydroxydopamine (6-OHDA) and MPTP induced Parkinson's models in Nrf2-/- and wild-type mice. Finally, we will examine Nrf2-mediated neuroprotection by infecting astrocyte cultures with adenovirus Nrf2 constructs and transplanting those cells into the striata of Nrf2-/- and wildtype mice after 6-OHDA or MPTP lesions. The specific aims of this proposal are: Specific Aim 1. Characterize Nrf2-dependent ARE activation by chemical activators and evaluate their neuroprotective potential in vitro. Specific Aim 2. Determine that chemical activators of the Nrf2-ARE pathway confer protection from the 6-OHDA and MPTP-induced nigrostriatal lesions and loss of dopaminergic (DA) neurons. Specific Aim 3. Determine that transplantation of Nrf2 overexpressing neural stem cells and/or astrocytes confers protection from 6-OHDA or MPTP-induced nigrostriatal lesions and loss of DA neurons.
The goal is to determine consequences perinatal TCDD exposure on
prostate development in the C57BL/6 mouse and elucidate the mechanisms
involved. Specific aims are to identify aberrant effects of TCDD on development
of ventral, dorsolateral, and anterior prostate and to determine using aryl
hydrocarbon receptor (AhR) knockout (AhRKO) mice if these effects require AhR.
The specific aims of the proposal are: 1) to determine critical periods for
producing TCDD effects and elucidate androgen-dependent mechanisms involved in
causing them. 2) to assess if TCDD acts directly on urogenital sinus (UGS) to
disrupt prostate development and if AhR is required in UGS mesenchyme or
epithelium to cause certain effects. 3) to evaluate if coexposure to a natural
AhR antagonist in human food, resveratrol, ameliorates TCDD disruption of
prostate development. 4) to identify TCDD-responsive genes in UGS and determine
which ones are involved in TCDD inhibition of prostate budding. 5)to determine
the long-term consequence of perinatal TCDD exposure on prostate size,
histology, and growth regulation in senescent mice. Hypotheses to be tested are
that perinatal TCDD exposure inhibits prostatic bud formation, ductal branching
morphogenesis, ductal canalization, luminal cell differentiation and prostatic
secretory function, and may alter normal age-related changes in androgen
dependence. The long-range goal is to identify genes that are the most
sensitive to TCDD exposure and whose altered expression may be critical for
producing particular aberrant prostate effects later in life. Genes implicated
in prostate development including those for proto-oncogenes, transcription
factors, homeobox genes, growth factors, and cell adhesion molecules, will be
assessed for differential expression along with other genes. The innovation in
this approach is that, for the first time, it may shed light on TCDD-induced
alterations in gene expression during fetal stage of development that cause
latent, adverse effects on the prostate. The human health significance is that
aberrant effects on prostate development are among the most sensitive and
highly reproducible responses to TCDD in animals. AhR and ARNT are expressed in
human fetal, benign hyperplastic, and malignant prostate, suggesting that human
prostate responds to TCDD. There is an extraordinarily high incidence of benign
prostate hyperplasia (BPH) and prostate cancer in humans, yet the function of
AhR signaling in human prostate remains unknown. The proposed research may
clarify the mechanisms by which TCDD disrupts early prostate development and
possibly prostate growth in senescence and provide insight into naturally
occurring chemicals in the human diet that may ameliorate the adverse
development effects of TCDD on the prostate.
DESCRIPTION (provided by applicant): The bronchial epithelium is a primary target of inhaled chromium(VI) compounds, which are known to cause serious pulmonary toxicity and carcinogenesis. The mechanisms underlying these effects are not clear. In cells, Cr(VI) exposure results in oxidative damage and genotoxic effects. The reduction of Cr(VI), which can generate reactive species [e.g. Cr(V), Cr(IV), hydroxyl radical (OH)], is required for its toxic effects. There are key differences between rodents and humans in Cr(VI) activation, and a normal human bronchial epithelium model for Cr(VI) toxicity is lacking. Our long-term objectives are to understand the mechanisms responsible for Cr(VI) activation in human lung. Human data indicate a significant role for microsomal enzymes, with a central role for cytochrome b5 (b5). The overriding hypothesis is that b5 contributes significantly to Cr(VI) activation in bronchial epithelium. An integration of cellular and in vitro approaches will be used to elucidate this role. The in vitro use of purified b5 in liposomes and electrochemical studies will examine the kinetics of Cr(VI) reduction to prove that b5 is the proximate electron donor. The generation of reactive species and their ability to mediate various forms of DNA damage will be determined quantitatively over time at various Cr(VI) concentrations. A kinetic ESR approach will be used to distinguish OH from other hydroxylating species. The nature of Cr(V)-DNA complexes will be determined by ESR, and the effect of this binding on subsequent DNA damage will be determined. The cytotoxicity of Cr(VI), and the link between reactive species formation by b5 and subsequent DNA damage will be examined in human bronchial epithelial cells expressing various levels of b5. Iron markedly stimulates Cr(VI) activation but a role for intracellular iron in Cr(VI) toxicity has not been explored. Reductive Cr(VI) activation, cytotoxicity, and DNA damage will be examined in cells whose iron stores have been depleted by a non-toxic extracellular iron chelator. These studies provide a detailed analysis of a previously unexplored mechanism of Cr(VI) activation in bronchial epithelium. They will provide a deeper understanding of the mechanisms underlying Cr(VI) toxicity in human lung, and will lead to new insights on the role of b5 in toxicant activation
Crisp Terms/Key Words: DNA damage, cytotoxicity, toxicology, respiratory epithelium, bronchus, respiratory toxin, toxicant interaction, free radical oxygen, cytochrome b5 reductase, chromium, iron metabolism, cytochrome, electron spin resonance spectroscopy, adduct, microsome
DESCRIPTION (provided by applicant)
The goal of this project is to develop a GPS-enabled multi-analyte sensor that can detect and measure exposure to trace gases present in air, specifically ozone, carbon monoxide and carbon dioxide gases. This sensor is based on the innovative exploitation of the anchoring of liquid crystals to a surface functionalized with metal ions. In preliminary studies we have demonstrated the ability to identify combinations of metals that selectively bind ozone and to design multi-arrays of metals that create fingerprints specific for functional groups. We also demonstrated the use of this new technology to rapidly (~ 10 seconds) measure exposure to ppb concentrations of an organophosphate compound. The sensing surfaces we created could be tuned to be reversible, were unaffected by humidity, and responded in a dose-dependent manner. These data strongly support the feasibility of PlatypusTM technology as the basis of a compact, multi-analyte, field deployable personal device for detection of exposure to trace gases. In this proposal we will develop a sensor subsystem and integrate it with GPS-enabled components that track and store information such as degree of exposure, time and duration of exposure and exact location of exposure events. Our intended product will allow simple uploading of the data to a computer and will be tested under field conditions. Such a product is needed to facilitate the understanding of human susceptibility to environmental exposures.
DESCRIPTION (provided by applicant): Quantitative monitoring of personal exposure to environmental toxins is currently limited by expense, inconvenience and lack of appropriate technology, yet such monitoring is needed for investigation of health effects caused by low dose, chronic exposure to compounds such as pesticides or industrial toxins. We have previously demonstrated that low concentrations of a semi-volatile organophosphate pesticide can be detected from a vapor phase by utilizing chemically functionalized, nanostructured surfaces and liquid crystals (Platypus( technology). What is needed, however, is a monitor that simultaneously measures exposure to multiple compounds. In this proposal, we will demonstrate the feasibility of developing Platypus( technology for the quantitative and simultaneous detection of two different classes of semi-volatile pesticides (carbamates and organophosphates) and for the discrimination of specific compounds within each class of pesticide. The technology developed in this project can be adapted for detection of other industrial or environmental contaminants that are semi-volatile, and thus it has the potential to serve as the basis for a broadly useful, convenient and inexpensive class of passive monitors for multi-compound personal exposure assessment and environmental monitoring. In this proposal, we are developing a small, inexpensive wearable device which will provide a measurement of an individual's exposure to multiple chemicals present in his or her environment over a period of a day to a few weeks. This data is essential for associating exposure to specific compounds with adverse affects on public health.
DESCRIPTION (provided by applicant): Orofacial clefts are among the most common birth defects in humans and are associated with significant life- long morbidity and mortality. There is considerable epidemiological evidence to suggest that the interaction of genes and the environment contributes to the pathophysiology of cleft palate (CP). In order to study these gene-environment interactions, CP in murine models can be induced with the pollutant 2, 3, 7, 8-tetrachloro- dibenzo-p-dioxin (dioxin). A locus, chemically mediated teratogenesis 1 (Cmt1), which is genetically linked to CP susceptibility was identified using this murine model. This proposal aims to identify genes involved in CP pathophysiology through both genetic and genomic approaches. First, Cmt1 will be physically mapped using interval-specific congenic mice and haplotype comparisons of common inbred mouse strains to identify the underlying gene(s) involved in modifying susceptibility. Genetic variants that affect gene expression are pre- dicted to underlie a majority of biological variation, including disease susceptibility. Therefore, as a second approach, temporal gene expression profiles will be generated to compare molecular palatogenesis in sensi- tive and resistant strains of mice. Since dioxin teratogenesis is mediated by the aryl hydrocarbon receptor (AHR), a ligand-activated transcription factor, it is likely that the molecular mechanism of CP induction involves alterations in gene expression. Therefore, differentially expressed AHR responsive genes will be identified throughout palatogenesis and the downstream effects on palate development will be characteri- zed.' Genes involved in dioxin-induced CP may be common to CP caused by a variety of genetic and/or environmental insults and point to a common pathophysiologic mechanism of clefting. The characterized gene expression profiles associated with mammalian CP could be used in the future to screen pharmaceuti- cals and environmental contaminants for teratogenic activity. Relevance: The goal of this proposal is to understand how genes of an individual interact with environmental exposures to cause cleft palate. This research can be used to test chemicals for toxic potential, as well as identify people at risk for cleft palate. By understanding the risk factors that can lead to cleft palate, treatment and prevention strategies can be developed.
DESCRIPTION (provided by applicant): Since congenital heart defects constitute an important health concern, and the relationship between exposure to environmental contaminants during early development and adult-onset of cardiovascular disease is not well understood, there is a clear need for the generation of models to explain the processes by which environmental contaminants contribute to cardiac malformation and heart failure. TCDD (2,3,7,8- tetrachlorodibenzo-p-dioxin) is an environmental contaminant known to pose significant health risks. For example, exposure to dioxins such as TCDD is associated with congenital heart disease. Research by the Peterson/Heideman group has revealed that the heart is a target organ for TCDD-induced developmental toxicity in zebrafish, and it's effects on the heart resemble hypoplastic left heart syndrome in humans. My preliminary results show for the first time in zebrafish that exposure to sublethal concentrations of TCDD during early development and juvenile stages results in cardiac toxicity that persists in adults. The overall goal of this work is to determine if persistent, heritable cardiac toxicity is induced by sublethal exposure to TCDD during early life stages. To accomplish this goal, I will use zebrafish to test three hypotheses. First, I will test the hypothesis that sublethal exposure to TCDD during embryonic development will cause endpoints of cardiac toxicity and changes in gene expression that are similar to those caused by lethal TCDD exposure. Information obtained from these studies will help us to understand the mechanisms by which early developmental exposure to TCDD induces hypoplastic heart syndrome and heart failure in zebrafish. Second, I will test the hypothesis that exposure to TCDD during early life and juvenile stages will result in cardiac toxicity that persists in adults. These results will enable us to develop models for correlating exposure to TCDD during early development with adult-onset of heart disease. Third, I will test the hypothesis that sublethal exposure to TCDD during early life and juvenile stages results in cardiac toxicity that is heritable, and that their offspring show reduced development, growth and survival associated with impaired cardiac health. I will use biochemical approaches to confirm that reduced health and survival of the F1 generation is not the result of TCDD exposure via maternal transfer. Results obtained will lay the foundation for determining whether TCDD can induce cardiac disease via epigenetic mechanisms. This work will significantly contribute to our understanding of how exposure to sublethal concentrations of TCDD during early life stages contributes to susceptibility to heart disease later in life.
DESCRIPTION (provided by applicant)
The proposed renewal of the Short-Term Minority Research Training Program at the University of Wisconsin-Madison Molecular and Environmental Toxicology Center will provide 8-10 weeks of laboratory research experience in molecular and environmental toxicology to four (4) minority undergraduates, in the summer generally between their junior and senior years. The summer program will continue to be an integral part of the pre-doctoral and postdoctoral training program currently funded by NIEHS in the Center, and is coordinated with other summer programs on the University of Wisconsin-Madison campus. The summer program's goals are as follows:
1. To give trainees the scientific and social experience of working in a top-class science laboratory at a major research University.
2. To provide a challenging research experience which will leave students with a record and sense of achievement.
3. To demonstrate to students that toxicology is an exciting area for research and can provide challenging careers.
4. To present toxicology as a scientific discipline of particular relevance to economically disadvantaged communities.
5. To provide students with an idea of what graduate education involves and how best to prepare during the remainder of their undergraduate years.
6. To promote the University of Wisconsin-Madison as an option for graduate training in toxicology.
Summer trainees will carry out a research project in the laboratory of one of 21 trainers, 20 of whom also participate in the graduate program in Molecular and Environmental Toxicology. The laboratory experience (about 40 hours per week) will be accompanied by weekly tutorial sessions in toxicology that will include a variety of speakers from the METC program (faculty, students, and postdoctoral fellows) who discuss a variety of contemporary issues in toxicological research. The students also will receive training in how to read and understand a research paper, how to prepare a research proposal, write a scientific report, and give a scientific presentation. This program will coordinate with other University of Wisconsin-Madison programs for undergraduate minorities, including a symposium at the Program's conclusion. Application forms and publicity for the Program will be sent out along with graduate school publicity to more than 1,300 schools (March 1 deadline) and will be posted on the Center's Web site and minority-network Web sites. Additional contacts will be made with schools where long-term linkages are being developed. Priority will be given to students who express interest in, and have the potential to complete, the University of Wisconsin-Madison Ph.D. Program. Following the summer experience, the investigators will provide support as trainees plan for graduate education and careers.
DESCRIPTION (provided by applicant)
The training program in cellular and molecular mechanisms of toxicity involves 26 trainers from the Molecular and Environmental Toxicology Center (METC), which also provides central administration. Participating faculty have outstanding training records and well-funded programs, covering a variety of research areas such as developmental toxicology, carcinogenesis, targetorgan toxicity and the roles of inflammation and immunosuppression in disease susceptibility. Graduate students in trainers' labs from METC and other Ph.D. graduate programs (M.D./Ph.D., Cancer Biology, Cellular and Molecular Pathology, Molecular and Cellular Pharmacology, Biomolecular Chemistry, and Pharmaceutical Sciences) will be considered for support for a period of two to three years. Postdoctoral trainees will be supported for two years, but will be required to submit an Individual NRSA application in the first six months. The selection of trainees and oversight of the grant will be carried out by the Program Director and a seven-member Training Grant Committee selected from the 26 trainers for three-year terms. . The Center and training program were reviewed in 2003 by an External Advisory Board, and will be reviewed again by a Board composed of nationally recognized toxicologists at the midpoint of the next funding cycle. This proposal seeks support for eight predoctoral and four postdoctoral positions. All predoctoral trainees take a 12-credit Training Grant core curriculum comprised of courses in basic mechanistic toxicology, the environment and human disease, research ethics and career development, and toxicology research seminar, in addition to courses required by their graduate programs. Pre- and postdoctoral trainees will also be mentored in grant writing and supervision of undergraduate students working in the trainers' labs. A preliminary examination qualifies predoctoral trainees for the final research phase and thesis examination, although laboratory research is continuous from the first semester. Predoctoral trainee progress is monitored by Research Advisory Committees for each trainee (five faculty mentors including at least three trainers) and by the Training Grant Committee. Postdoctoral training is facilitated by appointment of a Postdoctoral Career Advisory Committee (two faculty mentors in addition to a trainer) that will recommend selected didactic courses and collaborations. The recruitment of minorities to the Program is given high priority and is facilitated by a NIEHS-funded Summer Minority Research Program for undergraduates. The University provides two-year special fellowships for minority and disadvantaged students. Upon completion of the training program, graduates usually undertake a period of postdoctoral training, after which they are well qualified to assume responsible career positions in toxicology in academic, governmental, or other public or private research institutions, or industrial laboratories.
BACKGROUND
This is a competing continuation application for years 31 - 35 of support that requests 8 pre-doctoral students and 4 postdoctoral fellows. During the last funding period, the number of mentors has increased from 19 to 26 with the number of physician-scientist increasing from one to four and the number of veterinarian-scientists increasing from two to three. The focus of the research of the training faculty is in the areas of carcinogenesis, teratogenesis, target-organ toxicity and the roles of environmental exposures on the development of toxicity and/or disease. During the last funding period, the administrative infrastructure was moved so that it is now administered out of the School of Medicine and Public Health instead of the College of Agriculture and Life Sciences. In order to improve the quality of the candidate pool for traineeships, students from other graduate programs will be considered for support as long as they are working in the laboratories of one of the training faculty and are working on a project that is in accordance with the research focus outline by this application.
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