Abstract  The main purpose of this project was to investigate the flows and composition of the different fractions of household wastewater: greywater, urine, faeces and of solid biodegradable waste. Thus, the flows and compositions of all four fractions from 32 apartments with 79 inhabitants in the ecological collective building Gebers in Skarpnack, Stockholm, were measured continuously during 21 days, divided into three periods. From the four fractions a great number of samples were taken. The sampling of greywater was done continuously and flows proportionally and the collected amounts were mixed at the end of each period. Urine, Faeces and solid biodegradable waste were collected, and at the end of each period mixed and sampled. All fractions were subsequently analyzed for pH, DM, ash COD, BOD7, TOC, ammonia, nitrate, nitrite, Kjeldal nitrogen, total phosphorus and the elements K, Ca, Fe, Mg, Na, S, Ag, Al, B, Bi, Cd, Co, Cr, Cu, Hg, Mn, Mo, Ni, Pb, Pd, Pt,Rh, Sb, Se, Sn, Sr, Te, W and Zn. For urine and greywater, conductivity, suspended solids and phosphates were also measured. The greywater was additionally analyzed for the following organic compounds; PAH, PCB, phthalates, alkylphenol etoxilates, organotin compounds, brominated flame-retardants and LAS. A total amount of 80 selected organic compounds were investigated.

Abstract  Water resources investigations.

Abstract  The selected remedy includes the installation of a vertical barrier to facilitate containment of the Site 32 source area; extraction and treamtent of groundwater from within and below the vertical barrier to prevent migration of contaminants in the source area overburden; and excavation and off-site disposal of Site 36 metals- and VOC-contaminated soil. Extracted groundwater will be treated in the modified Site 32/36 treatment plant and will be discharged via off-site (on-base) subsurface recharge trenches or surface application. Because this remedy will result in hazardous substances remaining on-site, a periodic review will be conducted by the Air Force, U.S. Environmental Protection Agency (EPA), and NHDES to ensure that the remedy is providing adequate protection of human health and the environment. This review will be conducted at least once every 5 years as long as hazardous substances remain on-site above health-based cleanup levels.

Abstract  BIOSIS COPYRIGHT: BIOL ABS. RRM RESEARCH ARTICLE HUMAN FISH ALLIGATOR ALKYL PHENOL PHTHALATE BISPHENOL A DDT POLYCHLORINATED BIPHENYLS CHLOROORGANIC COMPOUNDS ESTROGEN INFERTILITY ECOLOGY TOXICITY

Abstract  DESCRIPTION (provided by applicant): The main objective of this application is to determine whether the xenoestrogenic substances bisphenol A (BPA) and butyl benzyl phthalate (BBP) play a role in the initiation of human breast cancer, and if so, whether this effect is mediated by epigenetic mechanisms. The proposed study is based on the growing concern that estrogenic environmental compounds that act as endocrine disrupting chemicals might have potential adverse effects on hormone-sensitive organs such as the breast. This concern is further fueled by evidence indicating that natural estrogens, namely 17 ?-estradiol (E2), are important factors in the initiation and progression of breast cancer. Therefore, the concern that BPA and BBP, which have estrogenic properties and are widely distributed in the environment, might also be carcinogenic for the human breast is well justified. For accomplishing these goals we will utilize our in vitro in vivo model in which we have demonstrated the carcinogenicity of E2 in the human breast epithelial cells MCF-10F. The utilization of this powerful and unique model will provide us a tool for exploring whether BPA and BBP have relevance in the initiation of breast cancer. Furthermore, we have found that the expression of E2-induced transformation phenotypes is associated with hyper or hypomethylation of genes controlling branching and ductulogenesis. These are the basis for our rationale to postulate that the xenoestrogens BPA and BBP can induce neoplastic transformation by behaving as epigenetic modulators inducing silencing of critical genes by hypermethylation and/or histone modification that lead to the initiation and progression of breast cancer. For this purpose, we propose the following specific aim: To determine whether the xenoestrogens BPA and BBP induce neoplastic transformation in human breast epithelial cells and whether the expression of transformation phenotypes is associated with epigenetic changes in genes controlling branching and ductulogenesis, and if this is the case, to determine if modifying their methylation status reverts the neoplastic process. To accomplish this aim we will use MCF-10F cells and primary cultures of human breast epithelial cells obtained from reduction mammoplasty. The cells will be treated with BPA and BBP using a protocol similar to that used for the treatment of these cells with E2, which will serve as a control. Methylation studies will be performed using Restriction Landmark Genomic Scanning (RLGS) and the identified genes will be further studied using methylation specific PCR (MSP) followed by confirmation of their expression and functional role. Altogether, these studies will provide first hand evidence on whether xenoestrogenic substances are able to induce neoplastic transformation in HBEC and that epigenetic mechanisms are involved in this process. Furthermore the manipulation of the methylated status and silencing of those epigenetically modified genes will provide not only an understanding of how these environmental contaminants are involved in breast cancer initiation but also will give us tools for developing preventive strategies to counteract their effect in the general population. RELEVANCE TO PUBLIC HEALTH: These studies will provide first hand evidence on whether xenoestrogenic substances like BPA and BBP are able to induce neoplastic transformation in HBEC and that epigenetic mechanisms are involved in this process. Furthermore the manipulation of the methylated status and silencing of those epigenetically modified genes will provide not only an understanding how these widely environmental contaminants are involved in breast cancer initiation but also for developing preventive strategies to counteract their effect in the general population.

Abstract  Abstract Not Provided.

Abstract  Description (provided by applicant): Children in America's inner cities are exposed to multiple known and potential neurodevelopmental toxicants. The overall goal of this project is to examine associations between exposures to EDs, pesticides and PCBs and growth and development of children of an already existing racially and ethnically diverse urban cohort of mothers and children. This project will continue to assess the effects of prenatal exposures to organophosphates (including chlorpyrifos), pyrethroids, and PCBs on growth and neurobehavioral development. As a new direction, the investigators plan to assess the impacts of potentially endocrine disrupting chemicals, specifically phthalates and bisphenol A on fetal, infant and childhood growth and development. Currently, they have at least 300 actively participating mother?infant pairs and will add another 78 mothers and infants from a community intervention project in the previous cycle of this grant. To assess environmental exposure, both stored and newly collected maternal and infant urinary samples will be used along with questionnaires. Tests to assess neurodevelopment include the Brazelton Neonatal Behavior Assessment Scale at birth, the Bayley Scales of Infant Development at ages 1 and 2 years, and several additional tests at ages 4, 6 and 7 years. Growth measures include indices of fetal growth (weight, head circumference and length at birth) as well as weight, head circumference, and length at ages 1, 2, 4, 6, and 7 years and percent body fat and lean mass at ages 4, 6, and 7 years. In collaboration with Project 3, the investigators will also assess possible modulating influences of polymorphisms and enzymatic activity involving paraoxonases, lipases and glucuronidases on these associations. The unique contribution of this study is that it assesses the effects on childhood growth and neurodevelopment of current multiple potential exposures to developmental toxicants among inner city, minority children.

Abstract  DESCRIPTION (provided by applicant): The phthalate ester, diethylhexylphthalate (DEHP), is a constituent chemical of many plastics, conferring their flexibility. Plastics are used ubiquitously in the United States and most industrialized countries and DEHP, which is not chemically bound to the plastic matrix, leaches out over time: human exposure is therefore widespread. Exposure to DEHP is associated with disruption of male sexual development and decreased fertility. Studies by Earl Gray and colleagues have shown that DEHP is anti-androgenic, and that its mechanism of male reproductive toxicity is not through direct interference at the level of the androgen receptor. The P.I. and others have shown that androgen secretion by rat Leydig cells is compromised after in vivo exposures to DEHP. This leads to the hypothesis providing the overall theme to the present application, that the endocrine disrupting properties of DEHP result from adverse effects on Leydig cell development with consequent decreases in androgen secretion. Consistent with this hypothesis, recent data from the P.l.'s lab demonstrate the existence of a critical window of sensitivity for impairment of Leydig cell steroidogenesis in younger animals. In the experiments described for this renewal application, we will analyze the potential for DEHP exposures: I. to suppress expression levels of steroidogenic factor-I, a key nuclear transcription factor that regulates differentiation of Leydig cells and pituitary gonadotropes, and decrease the action of Mullerian inhibiting substance, an SF-1 regulated growth factor that limits Leydig cell division; II. to inhibit proliferation of progenitor Leydig cells; and III. to delay acquisition of steroidogenic enzyme gene expression and steroidogenic capacity. Chronic postnatal exposures to DEHP induced increased levels of gonadotropic secretion by the pituitary and elevated serum testosterone and estradiol levels, which were associated with Leydig cell hyperplasia, a precursor to tumorigenesis. Formation of Leydig cell tumors is commonly observed in DEHP-treated rats. We will describe the time course of DEHP induced Leydig cell hyperplasia and analyze the role of LH, testosterone, and estradiol in the development of tumorigenesis. These studies will be the first to define the involvement of Leydig cells in the endocrine disrupting effects of DEHP. Our data will facilitate identification of biomarkers of exposure to environmental pollutants, and provide information to regulatory agencies in setting limits for the use of DEHP and phthalates in consumer products

Abstract  DESCRIPTION (provided by applicant): Epidemiological and experimental studies suggest that disruption of embryonic programming and gonadal development during human fetal life can result in testicular dysgenesis, manifested as undescended testis, hypospadias, poor semen quality, and testicular cancer. Preliminary data indicates that exposure of Leydig cells to low and environmentally relevant concentrations of di-(2-ethylhexyl) phthalate (DEHP) in vivo and mono-ethylhexyl phthalate (MEHP) in vitro altered the genomic and proteomic profile of the cells in a manner that parallels the inhibition of hormone-dependent steroid formation and the induction of Leydig cell hyperplasia. The central objective of this proposal is to gain greater understanding of the fetal basis of male reproductive disorders by elucidating the mechanisms by which exposure of the fetus to the environmental antiandrogen DEHP suppresses fetal testosterone production and later, testosterone and estradiol production by the adult. Our major goals are to identify the cellular targets and the molecular mechanisms underlying the responses of the fetus to DEHP, and to reveal the mechanisms by which effects on the fetus lead to pathologies of the male reproductive tract in the adult. The overarching hypothesis is that in utero exposures to DEHP suppress fetal testosterone production by direct effects on fetal Leydig cells and/or on the mesenchymal cells that are the precursors of adult Leydig cells, and by doing so, suppresses postnatal development and function of the adult Leydig cell population. We will test this hypothesis with the following specific aims: (1) identify the cellular and molecular targets of gestational DEHP in the fetal testis; (2) identify the mechanism(s) by which gestational exposure to DEHP results in reduced testosterone production by the fetal testis; and (3) determine the effects of fetal exposure to DEHP on the formation and function of the adult population of Leydig cells and its impact on testicular function in the immature and adult testis. We believe that these Aims will identify the molecular signaling pathways and characterize their role in mediating the hyperplasic and antiandrogenic effect of phthalates leading to testicular dysgenesis in the adult. Taken together the proposed studies will unveil the endocrine disruptor-sensitive steps in the steroidogenic pathway that are affected by this antiandrogen, and the mechanisms and consequences of endocrine disruption on the endocrine milieu of the adult.

Abstract  DESCRIPTION (provided by applicant): The proposed study will determine whether early childhood exposures to phthalates are associated with the development of current asthma and proallergic immunoglobulin (Ig) E production at ages five to seven and whether current exposures to phthalates are associated with augmented airway inflammation and diminished lung function at ages five to seven. The research will confirm or refute recent epidemiologic findings of associations between phthalate exposures and childhood asthma, atopy and reduced lung function. This prior research is limited by incomplete exposure and outcome measures. However, results from a pilot undertaken for the proposed study also support the hypothesis that phthalate exposures are risk factors in the development of asthma. The proposed study will be conducted among a cohort of 400 children who reside in minority communities in New York City. These communities experience some of the highest childhood asthma rates in the world. Phthalate exposures are also widespread. The research will be performed within the ongoing longitudinal birth cohort study being conducted by the Columbia Center for Children's Environmental Health (CCCEH). The CCCEH is evaluating the contribution of prenatal and postnatal exposures in the development of asthma and other health outcomes. Children are followed from pregnancy through age seven years. The proposed research is cost effective in that many of the required exposure and outcome measurements are already being gathered within the CCCEH cohort. These include detailed history of respiratory and allergic symptoms, asthma treatment and emergency room visits; lung function testing at age six; allergen levels in house dust samples collected from pregnancy through age five; and measurement of allergic sensitization (total and specific IgE measured at ages two, three, and five years). The phthalates will be measured in stored urine samples collected from the mother during pregnancy and from children at ages three and five years and in newly collected indoor air and urine samples at ages five to seven. Also at age five to seven years, exhaled nitric oxide will be measured as an indicator of airway inflammation and total and allergen-specific IgE levels will be determined. The case ascertainment of current asthma at ages five to seven will be made by a designated board- certified pediatric pulmonologist at the Morgan Stanley Children's Hospital of New York Presbyterian Hospital. Hypotheses to be tested are: (1) that early childhood exposures to the phthalates, as measured by metabolite levels in urine samples collected during pregnancy and at ages three and five years, predict current asthma at ages five to seven and production of IgE antibodies at age seven; and (2) after correcting for early phthalate exposure, current exposure as measured by levels of the parent compounds in indoor air samples and metabolites in urine samples collected at ages five to seven years, will be associated inversely with lung function and positively with airway inflammation at ages five to seven years.

Abstract  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.

Abstract  DESCRIPTION (provided by applicant): An apparent increase in the prevalence of human male reproductive tract abnormalities during the past half century have led Skakkebaek to articulate "testicular dysgenesis syndrome" as an explanation for this phenomenon. According to Skakkebaek, the observed abnormalities-including malformed external genitalia (hypospadias), undesecended testis (cryptorchidism), spermatogenic defects, and testis germ cell cancer- result from the alterations in the in utero and perinatal hormonal milieu causing disruption of sensitive male reproductive tract development events. Attention is focused on environmental endocrine disrupting chemicals as possible causes of this developmental disruption. Phthalates are ubiquitous environmental contaminants and endocrine disrupting chemicals. Rats exposed to di-(n-butyl)phthalate) (DBP) during critical in utero window of male reproductive tract development have altered gene expression and a number of reproductive tract abnormalities associated with testicular dysgenesis syndrome, including underdeveloped or absent reproductive organs, hypospadias, cryptorchidism and decreased sperm production. Although testis germ cell cancer has not been observed in this rat model, the fetal testes of DBP-exposed male rats contain dysplastic, multinucleated gonocytes. In this project, a new mouse model of gestational DBP-induced testicular dysgenesis is established. After characterizing alterations in gene expression in this genetically tractable species, p53-deficient mice will be used to allow DBP-induced dysplastic gonocytes to persist in the postnatal testis rather than degenerate and die. We expect these persistent dysplastic gonocytes to become transformed in the p53-deficient environment, resulting in tumorigenesis and the development of testis germ cell cancer. These goals will be pursued with the following working hypothesis as a guide: Disruption of mammalian in utero hormonal environment by phthalates produces a common spectrum of abnormalities across species, including a predilection to testicular carcinogenesis unmasked in p53-deficient mice.

Abstract  DESCRIPTION (provided by applicant): The goals of this study are 2-fold: 1) to characterize prenatal phthalate exposures among urban minority mothers and newborns using environmental and biologic monitoring; and 2) to examine effects of exposure on placental function, gestational age and fetal growth. The research is timely. Phthalates are widely used in consumer products and exposures are ubiquitous. 75% of the U.S. population is exposed; women receive higher exposures than men. This has implication for pregnancy as a number of phthalates are endocrine disrupters and have been shown experimentally to modulate steroidogenesis in the placenta, downregulate estradiol and testosterone levels and adversely affect fetal growth. Our pilot data in human populations also indicate that prenatal exposures are reducing gestational age. The proposed study is the first to characterize phthalate exposures specific to urban minorities and to assess endocrine disruption in the placenta and its implication for gestational age and fetal growth. The research is cost-effective in that it will be nested within a well-designed prospective cohort study being conducted by the Columbia Center for Children's Environmental Health. Costs of enrollment, collection and storage of environmental and biologic samples, questionnaires and medical record data are covered under the existing funding. Prenatal phthalate exposures will be characterized in 300 mother/newborn pairs using questionnaires, personal and indoor air monitoring and the measurement of phthalates in biologic samples collected from the mothers and newborns. Repeat measures in a subset will enable evaluation of temporal variability in exposure levels. The study will assess associations between prenatal phthalates, expression of genes involved in steroidogenesis, oxidative stress, and xenobiotic metabolism in the placenta, gestational age and infant birth weight, length and head circumference. The research brings together a collaborative team from Columbia and Harvard Universities, the Centers for Disease Control and Southwest Research Institute with expertise in molecular epidemiology, exposure assessment and health effects of phthalates.

Abstract  Germ cell apoptosis is the final common pathway of injury following exposure to toxicants that target the interacting cell types necessary for successful spermatogenesis. Model toxicants have been developed as functional probes of each of these interacting cell types: 2,5-hexanedione, carbendazim, and mono-(2-ethylhexyl)phthalate target Sertoli cells, ethane-1,2-dimethane sulfonate targets Leydig cells, and x-irradiation targets germ cells. Over the past quarter century, many labs have contributed to a rich database describing how each of these model toxicants acts one at a time to produce testicular injury. However, real world exposures, like those occurring at Superfund sites and Brownfields in Rhode Island, involve complex mixtures of hazardous chemicals. This project takes the next step toward mechanistic understanding of complex exposures by combining these model testicular toxicants in a novel co-exposure paradigm. 2,5-Hexanedione exposure is characterized by a 3-week prodromal phase followed by the rapid onset of Sertoli cell dysfunction and germ cell loss. In preliminary experiments, we show that early during 2,5-hexanedione exposure, the seminiferous epithelium is highly susceptible to co-exposure toxicity by carbendazim, a Sertoli cell toxicant with the same subcellular target as 2,5-hexanedione, even though each toxicant alone produces only a modest injury response. This 2,5-hexandeione-induced sensitization of the seminiferous epithelium is exploited by model toxicant co-exposure to test the following working hypothesis: the extent of co-exposure synergy following 2,5-hexanedione priming of the seminiferous epithelium depends upon targeting and shared molecular perturbations. We address this hypothesis by pursuing these Specific Aims: 1) Characterize the dose response and time dependence of 2,5-hexanedione-induced sensitization to carbendazim co-exposure 2) Determine the dependence of the co-exposure response to model toxicants in the 2,5-hexanedione-primed testis on cellular and subcellular targeting 3) Use gene chips to obtain molecular fingerprints of testicular sensitization and the model toxicant injury responses to predict co-exposure toxicity.

Abstract  DESCRIPTION (provided by applicant): The proposed study will determine whether early childhood exposures to phthalates are associated with the development of current asthma and proallergic immunoglobulin (Ig) E production at ages five to seven and whether current exposures to phthalates are associated with augmented airway inflammation and diminished lung function at ages five to seven. The research will confirm or refute recent epidemiologic findings of associations between phthalate exposures and childhood asthma, atopy and reduced lung function. This prior research is limited by incomplete exposure and outcome measures. However, results from a pilot undertaken for the proposed study also support the hypothesis that phthalate exposures are risk factors in the development of asthma. The proposed study will be conducted among a cohort of 400 children who reside in minority communities in New York City. These communities experience some of the highest childhood asthma rates in the world. Phthalate exposures are also widespread. The research will be performed within the ongoing longitudinal birth cohort study being conducted by the Columbia Center for Children's Environmental Health (CCCEH). The CCCEH is evaluating the contribution of prenatal and postnatal exposures in the development of asthma and other health outcomes. Children are followed from pregnancy through age seven years. The proposed research is cost effective in that many of the required exposure and outcome measurements are already being gathered within the CCCEH cohort. These include detailed history of respiratory and allergic symptoms, asthma treatment and emergency room visits; lung function testing at age six; allergen levels in house dust samples collected from pregnancy through age five; and measurement of allergic sensitization (total and specific IgE measured at ages two, three, and five years). The phthalates will be measured in stored urine samples collected from the mother during pregnancy and from children at ages three and five years and in newly collected indoor air and urine samples at ages five to seven. Also at age five to seven years, exhaled nitric oxide will be measured as an indicator of airway inflammation and total and allergen-specific IgE levels will be determined. The case ascertainment of current asthma at ages five to seven will be made by a designated board- certified pediatric pulmonologist at the Morgan Stanley Children's Hospital of New York Presbyterian Hospital. Hypotheses to be tested are: (1) that early childhood exposures to the phthalates, as measured by metabolite levels in urine samples collected during pregnancy and at ages three and five years, predict current asthma at ages five to seven and production of IgE antibodies at age seven; and (2) after correcting for early phthalate exposure, current exposure as measured by levels of the parent compounds in indoor air samples and metabolites in urine samples collected at ages five to seven years, will be associated inversely with lung function and positively with airway inflammation at ages five to seven years.

Abstract  DESCRIPTION (provided by applicant) Two classes of industrial chemicals with endocrine-disrupting capability-the phthalates and the alkylphenols-have become widely dispersed in the urban built environment, and significant levels of phthalates are now nearly ubiquitous in the bodies of Americans. Highest exposures occur in children and in minorities. Infants and children appear especially susceptible to disruptors, because of their disproportionately heavy exposures and the vulnerability of their still forming organs to any disruption of the hormonal signaling that irreversibly shapes early development. Yet little is known, either of children's pathways of exposure, or of the human developmental toxicity of EDs. To address these gaps, the Mount Sinai Center for Children's Environmental Health and Disease Prevention Research proposes, 1) to characterize the levels and sources of children's exposures to contemporary-use EDs in the urban built environment; 2) to study relationships between EDs and neurobehavioral development; 3) to study relationships among ED exposures, diet, physical activity, and somatic growth; 4) to characterize previously unexplored enzymatic polymorphisms that may modulate individual susceptibility to EDs; and 5) to develop and deploy culturally appropriate, evidence-based strategies in East Harlem to improve children's diets, increase physical activity, reduce obesity, reduce ED exposures, and promote good health. Project 1, the Community-based Prevention Research Project (CBPR), Growing Up Healthy in East Harlem, is built on a long-standing partnership with the East Harlem community. It will study levels and sources of urban children's exposures to EDs and assess relationships among ED exposures, diet, physical activity, obesity, and use of personal care products. Project 2, an ongoing prospective epidemiological study, will analyze new and previously banked biological samples to examine associations between pre- and postnatal exposures to EDs and growth and development in a cohort study of urban children. This project will also continue to assess the developmental effects in this cohort of early exposures to neurotoxicants-organophosphates, pyrethroids, PCBs, and lead-that have been its focus for the past 5 years. Project 3, a molecular genetic study, will assess gene-environment interactions that may influence individual susceptibility to EDs by identifying and characterizing polymorphisms and variations in expression levels of PON1, lipase, and UGT-glucuronyltransferase enzymes involved in ED metabolism. A new Community Outreach and Translation Core (COTC) will use scientific information from the Center to educate and empower community leaders in East Harlem and to inform policy makers and health professionals regionally and nationally about links between the urban environment and children's health. The Center will contain an Exposure Assessment Core that collaborates with the laboratories of the Center for Disease Control and Prevention (CDC) National Center for Environmental Health, a Biostatistics and Data Management Core and an Administration Core. The Center will support two new investigators in children's environmental research.

Abstract  DESCRIPTION (provided by applicant): The proposed study is a competing continuation of R01 ES09718, 'Environmental Organochlorines and semen quality'. There is scientific and public concern about whether several families of chemicals considered endocrinologically active, such as PCBs and phthalates, have adverse male reproductive health effects. Concern stems from studies showing that a majority of the U.S. general population are exposed to PCBs and phthalates, as well as animal and limited human studies suggesting possible associations of PCBs and phthalates with adverse reproductive health effects. In the proposed competing continuation, we will build upon our earlier work and more precisely define the exposure-response relationships we found between estrogenic and anti-estrogenic PCBs and alterations in semen parameters and reproductive hormones. We will extend our investigation to include measurements for hydroxylated metabolites of PCBs (OH-PCBs) since studies report that OH-PCBs are prevalent in human serum and may be more biologically active than the parent PCBs. The proposed new research direction on phthalates and male reproductive health evolved from a pilot study conducted in collaboration with the CDC. Long-standing toxicological data consistently shows that select phthalatesare male reproductive toxins. Leveraging the ongoing study on organochlorines and semen quality, we analyzed urine samples for phthalate monoesters from a sub-group of men. The results were suggestive of an association between select phthalates and alterations in semen parameters and reproductive hormones, as well as increased sperm DNA damage measured with the comet assay. The apoptosis DNA diffusion assay will be incorporated into the proposed study since the measurement of apoptosis is a necessary counterpart to the assessment of sperm DNA damage, as many cells with significant DNA damage are eliminated by apoptosis. To determine the optimal design for assessing exposure to phthalates, we conducted an exposure study on a sub-sample of men from the organochlorine and semen quality study. Each subject collected nine urine samples over three months. Variance components for between-subject and with-in subject, consisting of monthly and daily variances, were calculated. Based on our results, the optimal exposure assessment strategy for the proposed study is to collect three urine samples from all subjects on day 1, 2 and 31 of their study participation.

Abstract  Immunoglobulin (1g) variable regions are tumor associated antigens of B cell lymphomas and meyelomas. Vaccination with intact Ig has induced tumor specified immunity, but this approach has produced variable results and has led to the outgrowth of tumor variants. We hypothesize that the variable results seen may be due to the elicitation of a limited immune response to single or very restricted set of immunodominant epitopes when whole Ig is used as the vaccine. Our goal is therefor to define discrete VH peptides representing subdominant or cryptic epitopes from both the hyper variable and conserved framework regions to design a vaccination strategy that could provoke a broader response. In addition a vaccine that provokes protective immunity against epitopes within more conserved regions including framework regions of VH could be utilized for multiple patients. Our strategy is to identify peptides from he entire VH region with predicted binding affinities for H-2Kd, and test them for their ability to generate MHC-restricted CTL. Mice are immunized with dendritic cells pulsed with synthetic peptides, with dendritic cells transfected to express the selected peptides, or with dendritic cells transfected with the entire VH region. Our rational is that by using dendritic cells and focusing upon selected peptides it will be possible to generate CTL responses to areas of the VH that would otherwise be ignored due to the suppressive effects of more dominate epitopes. To test our hypothesis, antibody forming clones derived from the immune response of BALB/c mice to dextran and to the hapten phthalate have been immortalized as hybridomas and associated VH region peptides will be used to assess the specificity of the T cell repertoire and the hybridoma cells used as tumor models. The strengths of these two models are that (a) each hybidoma represents a dominant clonotype in the primary or secondary response, and (b) the Vh regions of these hybidomas have been sequenced and MHC binding peptides from these regions have been identified. While we expect to see tumor specific MHC-restricted CTL responses to both germ line and somatically mutated peptides an attempt being made to enhance the responses by linking peptides to heat shock proteins, by the local release of cytokines from biodegradable micro spheres and by treatment of dendritic cells with CpG-containing oligodeoxynucleotides. The efficacy of vaccination is evaluated in vitro and also tested in vivo by assessing the ability of immunized mice to withstand a tumor challenge or to prevent the outgrowth of tumor in remission. Finally, the effect of vaccination upon the normal B-cell repertoire expression is assessed.

Abstract  DESCRIPTION (Adapted from the applicant's abstract): The focus of this research project is on the mechanisms responsible for initiating testicular germ cells to undergo apoptosis after toxicant-induced Sertoli cell injury. Recent evidence suggests that exposure to environmental toxicants (organic chemicals, metals, and heat) is responsible for a decline in semen quality in men over the Last 50 years. However, despite this association of toxicant exposure and male infertility, Little is known of the mechanisms by which these agents cause a loss of germ cells. In this research project, the investigators will use mono-(2-ethyLhexyL) phthalate (MEHP), a widely characterized Sertoli cell toxicant, as our primary model agent to study the mechanisms initiating germ cells to undergo apoptosis. In the previous grant-funding period the investigators established that the Fas (CD95)- signaling pathway participates in the initiation of testicular germ cell apoptosis after MEHP-induced Sertoli cell injury. Recently, they made two exciting and critical observations: 1) The re-distribution of Fas in germ cell changes from a cytosolic to a membrane localization after MEHP exposure and, 2) young gld mice, that lack a functional form of FasL, are not sensitive to MEHP-induced apoptosis whereas adult gld mice are sensitive. These fundamental observations have led to the development of two working hypotheses. 1) Fas plays the dominant role in the initiation of MEHP- induced germ cell apoptosis, and, 2) p53 activation leads to the expression of Fas on the germ cell membrane and confers its sensitivity to apoptosis. The first specific aim of this proposal utilizes mutant mice that express either dysfunctional Fas (lpr mice) or FasL (gld mice) to directly examine the dependence of MEHP-mediated germ cell apoptosis on the cellular expression of Fas. In the second aim, the involvement of p53 in mediating the membrane distribution of Fas is tested both in vitro, using GC-2spd cells that express a temperature sensitive mutant of p53, or in vivo, using p53 null mice. In the Last two aims, the two hypotheses are further challenged by evaluating both the importance of the soluble form of FasL and the participation of alternative Fas-independent signaling mechanisms in the pathogenesis of MEHP-induced increases in germ cell apoptosis. This investigation of MEHP-stimulated germ cell apoptosis will provide fundamental insights into mechanisms regulating the sensitivity of germ cell to undergo apoptosis after chemical-induced testicular injury.

Abstract  DESCRIPTION (provided by applicant): The proposed study is a competing continuation of R01 ES09718, 'Environmental Organochlorines and semen quality'. There is scientific and public concern about whether several families of chemicals considered endocrinologically active, such as PCBs and phthalates, have adverse male reproductive health effects. Concern stems from studies showing that a majority of the U.S. general population are exposed to PCBs and phthalates, as well as animal and limited human studies suggesting possible associations of PCBs and phthalates with adverse reproductive health effects. In the proposed competing continuation, we will build upon our earlier work and more precisely define the exposure-response relationships we found between estrogenic and anti-estrogenic PCBs and alterations in semen parameters and reproductive hormones. We will extend our investigation to include measurements for hydroxylated metabolites of PCBs (OH-PCBs) since studies report that OH-PCBs are prevalent in human serum and may be more biologically active than the parent PCBs. The proposed new research direction on phthalates and male reproductive health evolved from a pilot study conducted in collaboration with the CDC. Long-standing toxicological data consistently shows that select phthalatesare male reproductive toxins. Leveraging the ongoing study on organochlorines and semen quality, we analyzed urine samples for phthalate monoesters from a sub-group of men. The results were suggestive of an association between select phthalates and alterations in semen parameters and reproductive hormones, as well as increased sperm DNA damage measured with the comet assay. The apoptosis DNA diffusion assay will be incorporated into the proposed study since the measurement of apoptosis is a necessary counterpart to the assessment of sperm DNA damage, as many cells with significant DNA damage are eliminated by apoptosis. To determine the optimal design for assessing exposure to phthalates, we conducted an exposure study on a sub-sample of men from the organochlorine and semen quality study. Each subject collected nine urine samples over three months. Variance components for between-subject and with-in subject, consisting of monthly and daily variances, were calculated. Based on our results, the optimal exposure assessment strategy for the proposed study is to collect three urine samples from all subjects on day 1, 2 and 31 of their study participation.

Abstract  DESCRIPTION (provided by applicant): Phthalates, widely used as plasticizers in food and biomedical packing, present a hazard to male reproductive health. DEHP, the most common phthalate, is metabolized to its active monoester, MEHP. In the testis, MEHP's primary testicular target is Sertoli cells (SC). However, germ cells (GC) undergo dramatic MEHP-induced changes such as detachment from SC and subsequent GC apoptosis. The long-term objective of this study is to elucidate the role of oxidative stress-induced GC mitochondrial dysfunction in MEHP toxicity and characterize the molecular targets involved. The specific aims to carry out this objective are to: (1) test the postulate that MEHP affects testicular expression of the antioxidant peroxiredoxins (Prx), (2) determine whether MEHP exposure leads to Prx3 inactivation in GC, but not in SC, (3) establish whether Prx3 inactivation leads to increased ROS generation and mitochondrial dysfunction in GC, but not in SC, and (4) characterize whether Prx3 plays a role in toxicant-mediated cytochrome c release from GC mitochondria. Mechanistic studies involving state of the art molecular cell biology techniques will be used to elucidate the role that mitochondrial dysfunction plays in phthalate-induced testicular toxicity and infertility.

Abstract  Description (provided by applicant): The overall goal of this molecular genetic project is to assess gene?environment interactions that may influence individual susceptibility to the EDs and developmental neurotoxicants that are the overall scientific theme of this Center. The plan is to meet this objective by identifying and characterizing polymorphisms and variations in expression levels of PON1, lipase and UGT-glucuronosyltransferase, all involved in the metabolism and detoxification of EDs and pesticides. The specific aims of this application are, Phthalates: 1) examine salivary lipase activity as a biomarker for conversion of phthalate diesters to phthalate monoesters; 2) determine expression levels of the three linked human lipase genes in human salivary glands; 3) determine racial/ethnic differences in the frequencies of the common polymorphisms in the salivary human lipase gene(s) and in salivary lipase activity; 4) examine salivary lipase activity in relation to urinary levels of phthalate metabolites; 5) examine salivary lipase polymorphisms and haplotypes in relation to urinary levels of phthalate metabolites; 6) obtain pilot data on the extent of glucuronidation of phthalate monoesters in urine; and 7) collaborate with the epidemiology project (Project 2) to examine association of lipase genotype and phenotype with child developmental outcomes. Bisphenol A: 1) genotype the common missense polymorphism in human UGT2B7 in our population of African-Americans, Caucasians and Hispanics and its association with urinary metabolites; 2) identify and assess common promoter, coding region and splice-site polymorphisms in human UGT2B7 in our African-Americans, Caucasians and Hispanics, establish the common haplotypes, and their association with exposure; and 3) collaborate with Project 2 to examine human UGT2B7 genotype/haplotype effects on developmental outcomes. Continuing PON1 phenotype?genotype studies: 1) determine high-density lipid (HDL) levels for the existing birth cohort and carry out a detailed genotype?phenotype reanalysis, 2) genotype and haplotype new members of the extended birth cohort for PON1 polymorphisms; and 3) collaborate with Project 2 to examine PON1 genotype and phenotype effects on developmental outcomes.

Abstract  DESCRIPTION (provided by applicant): The proposed study is a competing continuation of R01 ES09718, 'Environmental Organochlorines and semen quality'. There is scientific and public concern about whether several families of chemicals considered endocrinologically active, such as PCBs and phthalates, have adverse male reproductive health effects. Concern stems from studies showing that a majority of the U.S. general population are exposed to PCBs and phthalates, as well as animal and limited human studies suggesting possible associations of PCBs and phthalates with adverse reproductive health effects. In the proposed competing continuation, we will build upon our earlier work and more precisely define the exposure-response relationships we found between estrogenic and anti-estrogenic PCBs and alterations in semen parameters and reproductive hormones. We will extend our investigation to include measurements for hydroxylated metabolites of PCBs (OH-PCBs) since studies report that OH-PCBs are prevalent in human serum and may be more biologically active than the parent PCBs. The proposed new research direction on phthalates and male reproductive health evolved from a pilot study conducted in collaboration with the CDC. Long-standing toxicological data consistently shows that select phthalatesare male reproductive toxins. Leveraging the ongoing study on organochlorines and semen quality, we analyzed urine samples for phthalate monoesters from a sub-group of men. The results were suggestive of an association between select phthalates and alterations in semen parameters and reproductive hormones, as well as increased sperm DNA damage measured with the comet assay. The apoptosis DNA diffusion assay will be incorporated into the proposed study since the measurement of apoptosis is a necessary counterpart to the assessment of sperm DNA damage, as many cells with significant DNA damage are eliminated by apoptosis. To determine the optimal design for assessing exposure to phthalates, we conducted an exposure study on a sub-sample of men from the organochlorine and semen quality study. Each subject collected nine urine samples over three months. Variance components for between-subject and with-in subject, consisting of monthly and daily variances, were calculated. Based on our results, the optimal exposure assessment strategy for the proposed study is to collect three urine samples from all subjects on day 1, 2 and 31 of their study participation.

Abstract  DESCRIPTION (provided by applicant): Phthalates are ubiquitous environmental pollutants found in plastic and PVC-containing devices. Human exposure to phthalates poses a serious risk to testicular function. Hallmarks of phthalate induced testicular injury include disruption of Sertoli-germ cell adhesion and FSH-stimulated cAMP accumulation by Sertoli cells. Although the mechanism of phthalate induced testicular injury is not known, these data suggest that both G protein-coupled receptor signaling and cell-cell adhesion are early targets of phthalate injury.Flamingo proteins contain extracellular cadherin cell-cell adhesion domains coupled to seven transmembrane helices, thus indicating their involvement in both cell-cell adhesion and G protein mediated signal transduction. Three Flamingo homologs are expressed in rat testis at the mRNA level, and their expression pattern is affected by phthalate exposure. The mRNA expression of Flamingo 1 and 2 decreased to 70% of control 3 hours post exposure and continued to decline at later time points. This grant proposal investigates the mechanism of phthalate induced testicular injury by examining this hypothesis: Phthalates induce phosphorylation, internalization and degradation of Flamingo 1 and 2 protein leading to Sertoli-germ cell detachment and germ cell apoptosis.