Project 1: Cytokine Polymorphisms and Preterm Birth
Project 2: Functional ATM Polymorphisms
Project 3: The Sea Urchin Embryo as a Model for Developmental Susceptibility to Environmental Neurotoxins
Project 4: Dermal Exposure to 1,6-Hexamethylene Diisocyanate Spray Painting Operations
Project 5: Uterine Fibroids in Pregnancy
Project: Cytokine Polymorphisms and Preterm Birth
There is increasing evidence that infection and the inflammatory response may play a crucial role in the etiology of preterm birth (<37 wks). Intrauterine infection has been found to increase intra-amniotic and cervicovaginal fluid concentrations of inflammatory cytokines; and, both intrauterine infection and elevated inflammatory cytokine concentrations are associated with preterm birth. Consequently, it has been suggested that a woman’s own immune response to infection may precipitate preterm birth. Polymorphisms in the genes that encode the pro-inflammatory cytokines TNF-a and IL-1ß cause increased cytokine secretion in response to bacterial lipopolysaccharide (LPS) stimulation, resulting in higher inflammatory activity. Conversely, polymorphisms in cytokines IL-6 and IL-1Receptor antagonist increase anti-inflammatory activity. Many of these polymorphisms are relatively common in Caucasians (15%-47%), and have been associated with more severe outcomes in infectious and autoimmune diseases. In addition, the TNF-a (-307) polymorphism has been found to increase the risk of preterm birth in two relatively small studies. No other cytokine polymorphisms have been evaluated in relation to preterm birth.
We propose to examine the relationship between selected cytokine polymorphisms and risk of preterm birth in the large, well characterized, Pregnancy, Infection and Nutrition (PIN) cohort. DNA will be extracted from blood samples that have been obtained from 282 case and 618 non-case participants, and genotyping of three cytokine polymorphisms [TNF-a (-307), IL-1ß (-511), and IL-6 (-174)] will be performed. The main effect of individual polymorphisms and multiple polymorphisms on the risk of preterm birth will be examined. Finally, we will perform an exploratory analysis of the interaction between infection during pregnancy, cytokine polymorphisms, and preterm birth.
This project would provide a unique and efficient opportunity to examine genetic susceptibility factors for an important reproductive outcome. This preliminary research will permit a fuller exploration of a broader array of polymorphisms of other cytokines, other inflammation genes, and genes in other pathways. In addition, future work would include utilization of fetal DNA samples for genotyping. The project would engage members of the Developmental Susceptibility and Genetic Susceptibility Research Cores as well as utilize the High Throughput Genotyping and Biostatistics and Epidemiologic Methods Core Facilities.
Project: Functional ATM Polymorphisms
This project will determine whether certain missense mutations in ATM produce a trait of G2-irradiation chromosomal hypersensitivity. A case-control study is underway at UNC-CH in which peripheral blood lymphocytes are tested for mutations in ATM and chromosomal hypersensitivity following treatment with ionizing radiation in G2. Cases represent women with newly diagnosed breast cancer and controls represent hospital patients matched for age and race but without cancer. To date ATM mutations and chromosomal hypersensitivity have been detected in both cases and controls. The frequency of the traits is increased about three-fold in cases relative to controls suggesting that ATM mutations and chromosomal hypersensitivity predispose to development of breast cancer. Several patient samples have been identified which display both mutations in ATM and chromosomal hypersensitivity. To prove that the trait of chromosomal hypersensitivity seen in these samples was a consequence of the mutation in ATM, a functional assay must be developed. We propose to clone ATM cDNA into a retroviral vector for efficient, stable transduction and expression of ATM in ATM-null cell lines. Expression of ATM in AT cells should restore DNA damage checkpoint functions and reduce chromosomal sensitivity to irradiation. Having a system in which ATM function can be demonstrated by restoration of checkpoint function and chromosomal repair, we will test the effect of introducing specific mutations in the ATM cDNA by site-directed mutagenesis. Missense mutations that were seen in lymphocytes with chromosomal hypersensitivity will be transduced alone or in combination with wildtype ATM. We will determine whether the missense mutations reduce the ability of ATM to enforce cell cycle checkpoints and enhance repair of chromatid breaks, and whether the mutations can override the function of wildtype ATM (dominant negative). This effort will help to define functional polymorphisms in a DNA repair gene.
Project: The Sea Urchin Embryo as a Model for Developmental Susceptibility to Environmental Neurotoxins
The purpose of the proposed project is to develop an animal model for in vivo screening of environmental neurotoxicants, using the early sea urchin embryo. The proposed studies will investigate mechanisms underlying embryotoxic effects of the organophosphate insecticide, chlorpyrifos (cpf). We aim to demonstrate that this invertebrate model allows identification of cellular targets and cellular/molecular mechanisms of cpf embryotoxicity that are relevant to neurotoxic effects of cpf on mammalian brain development. The Specific Aims are designed to test the hypothesis that embryotoxic effects of cpf on early sea urchin embryos are due to its ability to significantly increase intracellular calcium (Ca2+) by activation of nACh receptors (nAChR) and Ca2+ channels. We will determine whether the protective actions of lipophilic acetylcholine analogs, Ca2+ channel blockers or nAChR antagonists against embryotoxic effects of cpf involve prevention of increased intracellular Ca2+. Specifc Aim 1 will determine whether cellular substrates for chlopyrifos (cpf) embryotoxicity during vulnerable periods of early sea urchin development involve increased intracellular Ca2+. Specific Aim 2 will determine whether protective actions of lipophilic acetylcholine analogs, Ca2+ channel blockers or nAChR antagonists are mediated by their ability to block cpf-induced increases in intracellular Ca2+. These studies will provide clues as to possible cellular/molecular mechanisms of cpf embryotoxicity and provide evidence regarding the relevance of these mechanisms to cpf actions on mammalian brain development Future studies will utilize this information to delve deeper into cellular/molecular mechanisms. Ultimately, this simple animal model could provide the opportunity for development of pharmacologic agents useful for prevention of neurotoxicant effects on pregnancy outcome and brain development.
Project: Dermal Exposure to 1,6-Hexamethylene Diisocyanate Spray Painting Operations
Development and application of monitoring methods for measurement of 1,6-hexamethylene diisocyanate (HDI) exposure to spray painters are required to estimate the risk for dermal sensitization and development of HDI induced occupational asthma. The importance of this problem and the availability of a well-defined “at-risk” study population warrant the comprehensive investigation of multiple routes of HDI exposure. We will recruit 15 spray painters from an U.S. Marine/Navy base located in North Carolina for this pilot project. Personal breathing-zone exposure to HDI will be measured using the Iso-CheckT sampler. Dermal exposure will be measured using a non-invasive method for tape-stripping skin (stratum corneum) and quantification by analytical chemistry. Urine samples will be collected for analysis of 1,6-hexamethylene diamine (HDA) as a marker for exposure and systemic absorption. Together, these data will allow us to correlate dermal exposure to systemic exposure and to determine the significance of dermal exposure. In addition, we will synthesize the S-cysteinyl-(6-aminohexamethylene)carbamyl conjugate of the keratin-1 (KRT1) and keratin-10 (KRT10) fragments with the structures Gly3ArgPheSer2CysGly2 and Gly4CysGly5, respectively. If successful, these conjugates will be used to develop an enzyme-linked immunosorbent assay (ELISA) to determine cysteinyl-KRT1 and -KRT10 protein adducts as biomarkers of exposure to HDI in a future study. ELISA will allow sensitive detection of HDI-adducted keratin as a biomarker to measure HDI absorption and the potential systemic bioavailability from the skin. Correlation between the biologically available dose and the dose measured in the biological specimen is critical to developing an understanding of the role of isocyanate exposure through the skin. The results obtained with this study will provide us essential preliminary data for a development of comprehensive research proposal to investigate the relationship between dermal exposure, sensitization, and potential development of occupational asthma in this “at-risk” population.
Project: Uterine Fibroids in Pregnancy
Uterine fibroids, or leiomyomata, are benign smooth muscle and connective tissue tumors. They are the most common pelvic neoplasm in women, affecting more than 20% of reproductive age women. Physiologic changes of pregnancy favor fibroid growth, however individual women may experience regression, stability, or growth of fibroids during gestation. Little is known about the natural history and mediators of growth of fibroids; even less is known about their etiology. We are interested in exploring the relationship between exposure to phthalates and presence and behavior of fibroids during pregnancy. Phthalates are ubiquitous man-made chemicals. Specific phthalates have hormonal activity in animal models and in vitro; including acting as endocrine disruptors. Routes of exposure include inhalation and dermal absorption from products such as perfumes, hairspray, soaps, skin care products, nail polish, and other cosmetics. It has recently become feasible to measure urine metabolites of common phthalates. This has allowed new research on the health effects of phthalates in humans and interest in methods to assess phthalate exposure in large-scale studies. Fibroids and phthalates are stated research priorities of multiple federal funding agencies. Our team proposes to lay a foundation for future work on phthalates and fibroids by: (1) developing an instrument to relate self-report of product use and other phthalate exposures to urinary phthalate levels; (2) obtaining self-report of use of products likely to contain phthalates from 100 women with, and 100 women without fibroids at baseline; (3) conducting follow-up ultrasounds at 22-24 weeks gestation for 40 women with fibroids to measure their fibroids; (4) measuring urinary phthalate levels for the first 30 women with fibroids who have follow-up ultrasounds and for 20 women without fibroids. This work will provide the requisite preliminary data and methodologic experience to guide continued research on fibroids, phthalates, and their potential influence on reproductive health outcomes.
Funded by NIEHS Grant # P30 ES010126