Development of a stably transfected cell line to screen for potential endocrine disrupting chemicals [abstract]

Abstract only availableIntro Estrogens are steroid hormones that diffuse across cell membranes to interact with estrogen receptors (ER) in the nucleus. An estrogen-bound ER acts as a transcription factor to regulate gene transcription in a cell. Synthetic chemicals with estrogenic activity (called xenoestrogens) also interact with the estrogen receptor (ER), interfering with the body's delicate balance of hormones. There is a great need to characterize these chemicals as well as screen for until now unknown xenoestrogens. Methods We developed a stably transfected MCF-7 human breast cancer cell line that, in combination with reporter gene assays, will aid in screening for and characterization of these chemicals. MCF-7 cells were transfected with DNA containing two copies of an estrogen response element (ERE) from the promoter of the vitellogenin gene. The ERE was linked to either LacZ or the firefly luciferase gene (both reporter genes). When a chemical interacts with the ER, the ER will associate with the ERE to begin the transcription of the reporter gene that will encodes for a quantifiable protein. Cells were co-transfected with a plasmid containing the neomycin gene, conferring resistance to the antibiotic G418 to allow for selection of stable transformants. Following transfection, cells were treated with G418 and grown for 18-25 days until colonies formed. Colonies were selected and a dose response of estradiol, the body's most common estrogen, was performed on the clones. Those populations showing the greatest sensitivity to estrogen were chosen for future use. Ongoing Studies Future experiments will include using reporter gene assays to screen common chemicals, such as the pesticide carbaryl, for estrogenic activity both alone and in environmentally relevant combinations of other xenoestrogens. Secondly, bioassays on clinical samples of peritoneal fluid from women with an estrogen-dependent disease will be performed and compared to control samples to determine total estrogenic activity.Life Sciences Undergraduate Research Opportunity Progra

Effects of prenatal exposure to xenobiotic estrogen and the development of endometriosis in adulthood

Abstract only availableEndometriosis is an estrogen-dependent disease that affects millions of women worldwide, causing pain and infertility. While it is known that retrograde menstruation places endometrial tissue in the peritoneal cavity, it is unclear why it invades and proliferates in women with endometriosis. Studies have shown that other hormone-dependent diseases have a fetal basis (e.g. breast cancer), suggesting that the presence of different hormones before birth may alter the incidence of endometriosis in adulthood. For example, women whose mothers took the synthetic estrogen diethylstilbestrol (DES) during pregnancy had an eighty percent increased incidence of endometriosis. Thus, our hypothesis is that prenatal exposure to xenobiotic estrogen will increase the severity of endometriosis in adulthood in a mouse model of surgically-induced endometriosis. To test this hypothesis, mice were time mated and dosed with vehicle control, 100 ng/kg DES or 10,000 ng/kg DES from days 11-17 of gestation. Surgical induction of endometriosis was performed in adulthood by autotransplantation of one uterine horm. The horn was removed, opened, divided into three pieces, and sutured to the arterial cascade of the intestinal mesentery. The implants became vascularized and formed endometriotic lesions. The mice were then collected at 2 or 4 weeks post-surgery, and the following endpoints were measured: 1) uterine weight; 2) implant size; and 3) implant weight. Additionally, implants were set aside for further analysis of 1) histology; 2) estrogen receptor indicator reporter gene activity; and 3) endometriosis-related gene expression. At the conclusion of this ongoing study, we expect to show whether there is an estrogen-mediated fetal component to endometriosis.Life Sciences Undergraduate Research Opportunity Progra

Peptide profiling: Correlating estrogen receptor conformation with biological response

Abstract only availableChemicals found in the environment have been found to behave like the body's natural estrogen, estradiol. These exogenous estrogen-mimicking compounds have been termed xenoestrogens. Both estradiol and xenoestrogens can bind two estrogen receptors (ERs), ER alpha and ER beta, to elicit biological responses. The receptors are ligand inducible transcription factors that exhibit unique biological actions. While estradiol binds both receptors equally, some xenoestrogens have been shown to bind ER beta preferentially. When the ER is bound, the ligand induces a unique ER shape and in turn causes an array of tissue-specific biological responses. For example, the ligand tamoxifen, a commonly used breast cancer pharmaceutical, exhibits an ER antagonist response in the breast and an ER agonist response in the bone. This dual ligand quality characterizes what is now known as a selective estrogen receptor modulator (SERM). Peptide profiling, a novel ER ligand screening assay, is a method that can potentially identify SERMS by correlating in vitro ER conformation with in vivo biological response. Each ligand is screened using a two-hybrid fusion protein reporter gene assay. Upon ligand binding, the ER assumes a conformation; with this induced shape, some ER-interacting peptides will be able to bind while others will not. After screening a ligand against a library of fifteen different peptides, a unique peptide profile will figuratively illustrate the induced ER conformation. Eight xenoestrogens were screened in this experiment: estradiol, a natural physiological estrogen; resveratrol and genistein, two phytoestrogens; MPP, bisphenol A, and 4-hydroxytamoxifen, all synthetic estrogens; α-endosulfan and methoxychlor, both insecticides used on crops. Each ligand was found to have a unique peptide profile and, implicitly, a distinct ER conformation. The next step will be to determine each ligand's tissue specific activity and identify the unique peptide fingerprint that predicts its in vivo biological response. By correlating a ligand's tissue specific estrogenic activity with its unique ER conformation, peptide profiling will not only further elucidate tissue-specific ER activity differences but could also be used as a high-throughput screening tool for other potential environmental xenoestrogens and identify novel therapeutic SERMs.Life Sciences Undergraduate Research Opportunity Progra

Neonatal exposure to xenobiotic estrogen may alter the adult immune response and exacerbate endometriosis in mice [abstract]

Abstract only availableEndometriosis is a common medical condition affecting 5-10% of women worldwide and often results in severe cramps, pelvic pain, and infertility. The condition occurs when endometrial tissue, which escapes into the peritoneal cavity via retrograde menstruation, adheres to peritoneal cavity tissues and causes irritated, inflamed lesions. Studies have suggested that the risk of developing endometriosis increases in women who have been exposed to xenobiotic (foreign to the body) estrogens during development. This could be due to developmental programming of the peritoneal environment, and specifically, an altered immune function within this environment. Therefore, it is our hypothesis that developmental programming by xenoestrogens alters the immune response to shed endometrial tissue and exacerbates endometriosis. To better understand the role of xenoestrogens in immune programming, we are conducting our studies using a mouse model of surgically induced endometriosis. In particular, we are concentrating on two major aspects of immunity: 1) the presence of immune cells and 2) the function of those cells. Our study of the former is being largely performed using methods of immunohistochemistry (IHC). IHC allows us to quantify the macrophages present in the peritoneal fluid of experimental mice (exposed to diethylstilbestrol) versus control mice (no xenoestrogen exposure). In order to study our second focus, immune cell function, we are using a cytokine antibody array to determine the relative cytokine concentrations in the peritoneal fluid samples. By identifying the degree to which certain cytokine concentrations differ, we hope to better understand the effect of xenoestrogen exposure on immune cell function.Life Sciences Undergraduate Research Opportunity Progra

The Waldschmidt constant for squarefree monomial ideals

Given a squarefree monomial ideal $I \subseteq R =k[x_1,\ldots,x_n]$, we show that $\widehat\alpha(I)$, the Waldschmidt constant of $I$, can be expressed as the optimal solution to a linear program constructed from the primary decomposition of $I$. By applying results from fractional graph theory, we can then express $\widehat\alpha(I)$ in terms of the fractional chromatic number of a hypergraph also constructed from the primary decomposition of $I$. Moreover, expressing $\widehat\alpha(I)$ as the solution to a linear program enables us to prove a Chudnovsky-like lower bound on $\widehat\alpha(I)$, thus verifying a conjecture of Cooper-Embree-H\`a-Hoefel for monomial ideals in the squarefree case. As an application, we compute the Waldschmidt constant and the resurgence for some families of squarefree monomial ideals. For example, we determine both constants for unions of general linear subspaces of $\mathbb{P}^n$ with few components compared to $n$, and we find the Waldschmidt constant for the Stanley-Reisner ideal of a uniform matroid.Comment: 26 pages. This project was started at the Mathematisches Forschungsinstitut Oberwolfach (MFO) as part of the mini-workshop "Ideals of Linear Subspaces, Their Symbolic Powers and Waring Problems" held in February 2015. Comments are welcome. Revised version corrects some typos, updates the references, and clarifies some hypotheses. To appear in the Journal of Algebraic Combinatoric

Preconceptional, Gestational, and Lactational Exposure to an Unconventional Oil and Gas Chemical Mixture Alters Energy Expenditure in Adult Female Mice

Previous studies conducted in our laboratory have found altered adult health outcomes in animals with prenatal exposure to environmentally relevant levels of unconventional oil and gas (UOG) chemicals with endocrine-disrupting activity. This study aimed to examine potential metabolic health outcomes following a preconception, prenatal and postnatal exposure to a mixture of 23 UOG chemicals. Prior to mating and from gestation day 1 to postnatal day 21, C57BL/6J mice were developmentally exposed to a laboratory-created mixture of 23 UOG chemicals in maternal drinking water. Body composition, spontaneous activity, energy expenditure, and glucose tolerance were evaluated in 7-month-old female offspring. Neither body weight nor body composition differed in 7-month female mice. However, females exposed to 1.5 and 150 μg/kg/day UOG mix had lower total and resting energy expenditure within the dark cycle. In the light cycle, the 1,500 μg//kg/day group had lower total energy expenditure and the 1.5 μg/kg/day group had lower resting energy expenditure. Females exposed to the 150 μg/kg/day group had lower spontaneous activity in the dark cycle, and females exposed to the 1,500 μg/kg/day group had lower activity in the light cycle. This study reports for the first time that developmental exposure to a mixture of 23 UOG chemicals alters energy expenditure and spontaneous activity in adult female mice

Parma consensus statement on metabolic disruptors

A multidisciplinary group of experts gathered in Parma Italy for a workshop hosted by the University of Parma, May 16–18, 2014 to address concerns about the potential relationship between environmental metabolic disrupting chemicals, obesity and related metabolic disorders. The objectives of the workshop were to: 1. Review findings related to the role of environmental chemicals, referred to as “metabolic disruptors”, in obesity and metabolic syndrome with special attention to recent discoveries from animal model and epidemiology studies; 2. Identify conclusions that could be drawn with confidence from existing animal and human data; 3. Develop predictions based on current data; and 4. Identify critical knowledge gaps and areas of uncertainty. The consensus statements are intended to aid in expanding understanding of the role of metabolic disruptors in the obesity and metabolic disease epidemics, to move the field forward by assessing the current state of the science and to identify research needs on the role of environmental chemical exposures in these diseases. We propose broadening the definition of obesogens to that of metabolic disruptors, to encompass chemicals that play a role in altered susceptibility to obesity, diabetes and related metabolic disorders including metabolic syndrome

Global estimates of mortality associated with long-term exposure to outdoor fine particulate matter.

Exposure to ambient fine particulate matter (PM2.5) is a major global health concern. Quantitative estimates of attributable mortality are based on disease-specific hazard ratio models that incorporate risk information from multiple PM2.5 sources (outdoor and indoor air pollution from use of solid fuels and secondhand and active smoking), requiring assumptions about equivalent exposure and toxicity. We relax these contentious assumptions by constructing a PM2.5-mortality hazard ratio function based only on cohort studies of outdoor air pollution that covers the global exposure range. We modeled the shape of the association between PM2.5 and nonaccidental mortality using data from 41 cohorts from 16 countries-the Global Exposure Mortality Model (GEMM). We then constructed GEMMs for five specific causes of death examined by the global burden of disease (GBD). The GEMM predicts 8.9 million [95% confidence interval (CI): 7.5-10.3] deaths in 2015, a figure 30% larger than that predicted by the sum of deaths among the five specific causes (6.9; 95% CI: 4.9-8.5) and 120% larger than the risk function used in the GBD (4.0; 95% CI: 3.3-4.8). Differences between the GEMM and GBD risk functions are larger for a 20% reduction in concentrations, with the GEMM predicting 220% higher excess deaths. These results suggest that PM2.5 exposure may be related to additional causes of death than the five considered by the GBD and that incorporation of risk information from other, nonoutdoor, particle sources leads to underestimation of disease burden, especially at higher concentrations

The Microphenotron: a robotic miniaturized plant phenotyping platform with diverse applications in chemical biology

Background Chemical genetics provides a powerful alternative to conventional genetics for understanding gene function. However, its application to plants has been limited by the lack of a technology that allows detailed phenotyping of whole-seedling development in the context of a high-throughput chemical screen. We have therefore sought to develop an automated micro-phenotyping platform that would allow both root and shoot development to be monitored under conditions where the phenotypic effects of large numbers of small molecules can be assessed. Results The ‘Microphenotron’ platform uses 96-well microtitre plates to deliver chemical treatments to seedlings of Arabidopsis thaliana L. and is based around four components: (a) the ‘Phytostrip’, a novel seedling growth device that enables chemical treatments to be combined with the automated capture of images of developing roots and shoots; (b) an illuminated robotic platform that uses a commercially available robotic manipulator to capture images of developing shoots and roots; (c) software to control the sequence of robotic movements and integrate these with the image capture process; (d) purpose-made image analysis software for automated extraction of quantitative phenotypic data. Imaging of each plate (representing 80 separate assays) takes 4 min and can easily be performed daily for time-course studies. As currently configured, the Microphenotron has a capacity of 54 microtitre plates in a growth room footprint of 2.1 m², giving a potential throughput of up to 4320 chemical treatments in a typical 10 days experiment. The Microphenotron has been validated by using it to screen a collection of 800 natural compounds for qualitative effects on root development and to perform a quantitative analysis of the effects of a range of concentrations of nitrate and ammonium on seedling development. Conclusions The Microphenotron is an automated screening platform that for the first time is able to combine large numbers of individual chemical treatments with a detailed analysis of whole-seedling development, and particularly root system development. The Microphenotron should provide a powerful new tool for chemical genetics and for wider chemical biology applications, including the development of natural and synthetic chemical products for improved agricultural sustainability