Childhood obesity is a global epidemic with costly comorbidities. In the US, the rate of obesity has tripled over the past 40 years. Diet, physical activity, and genetics do not explain all of the variability in weight, and moreover can be difficut to modify. Identifying environmental triggers and feasible public health interventions is therefore a policy imperative. Prior data from this team has suggested an association of prenatal exposure to air pollution with maternal hyperglycemia and reduced fetal growth. Paradoxically we have also demonstrated an association between greater prenatal traffic-related pollution and excess infant weight gain after birth, leading to higher risk for obesity in infancy. With the guidance of my mentoring team, during this 5 year K23 career development award, the Candidate will leverage data from two prospective longitudinal cohorts of mothers and children (Project Viva, n=2,128 and the New Hampshire Birth Cohort Study (NHBCS), n=1,500) to extend this prior work through three inter-related projects. The Principal Investigator will: (1) examine prenatal exposure to ambient air pollution as a risk factor for rapid weight trajectories throughout childhood and central fat accrual in mid-childhood. (2) consider the relationship of prenatal air pollution exposure with obesity-associated hormonal biomarkers, including leptin, adiponectin, and insulin resistance in childhood. (3) characterize exposures and habits related to indoor sources of air pollution in rural New England and estimate the extent to which indoor wood burning during pregnancy is associated with maternal hyperglycemia, fetal growth, and offspring weight gain in early childhood. The training plan proposed will build upon my clinical training in pediatric endocrinology and basic knowledge of biostatistics and epidemiology to provide formal training in (1) environmental exposure assessment, (2) statistical techniques necessary to analyze complex exposure-response relationships, and (3) the practical skills necessary to lead a research team. The Candidate will leverage the wealth of resources available at Boston Children's Hospital, the Department of Population Medicine (Harvard Medical School and Harvard Pilgrim Health Care), the Harvard School of Public Health, and the Dartmouth Children's Environmental Health and Disease Prevention Research Center to conduct the proposed analyses. These studies and training will lay the necessary scientific framework to launch her career as an independent physician researcher studying the impact of environmental toxicants on children's health, with a focus on obesity and insulin resistance.

Cardiovascular disease (CVD) is the leading cause of morbidity and mortality worldwide and evidence has suggested that exposure to environmental toxicants can increase CVD risk. Recently, high levels of arsenic (As), a leading environmental contaminant of concern have been associated with increases in CVD incidence, mortality, blood pressure, and systemic inflammation. Arsenic's effects at lower exposure levels are uncertain and studies are limited, but exposure at these low levels may be more widespread than previously thought, as data suggest that both water and dietary sources may contribute to overall As exposure in adults and children. In this study, the candidate proposes to take a life course approach to examining the effects of a wider range of As exposure levels on CVD risk in both adults and children and identify preclinical indicators and biomarkers of susceptibility for CVD. In the K99 mentored phase, using existing data from two adult populations ranging in levels of As exposure, the candidate will explore the relationship between As exposure and biomarkers of CVD risk, while gaining expertise in molecular epidemiology. The candidate will test archived blood and urine samples from a U.S. population to examine the relationship between low level As exposure and markers of systemic inflammation, oxidative stress and endothelial function, which have been related to high levels of As exposure among adults. In a more highly exposed Bangladeshi population, the candidate will analyze a set of potential genetic susceptibility loci in relation to As exposure and their impact on blood pressure measurements over time. She will also advance her knowledge of molecular and cardiovascular disease epidemiology through formal coursework, workshops and guidance from a diverse advisory committee of respected researchers. In the R00 independent phase, she will apply her findings to initiate a new line of investigation testing whether in utero and/or early life As exposure influences early indicators of CVD risk in two populations of children, as pregnancy and early childhood are particularly vulnerable periods when environmental insults may impact development and thus later risk of disease. In conjunction with the Health Effects of Arsenic Longitudinal Study in Bangladesh and the New Hampshire Birth Cohort Study, the candidate will implement two parallel investigations of early indicators of CVD risk among five-year-old children exposed to As. She will test whether non-invasive measures of endothelial function are associated with varying levels of in utero and/or early childhood As exposure in these two populations, as subclinical effects of As on CVD risk may occur far in advance of clinical presentation. Developing insight into the mechanisms underlying As-related CVD and identifying biomarkers of CVD risk across life stages as they relate to As exposure, will help to define the contribution of environmental As exposure to different phases of disease, from initiation to clinical presentation. With this proposed study, the candidate is positioned to take advantage of existing resources to in order to develop independent, yet complementary projects, designed to help to fill critical gaps in our understanding of the lifelong cardiovascular health impacts of a range of As exposures levels.

There is growing evidence that lifelong health, including mental health, is particularly shaped by the environment experienced during the in utero developmental period. The intrauterine environment is influenced by a complex variety of factors, including maternal lifestyle, environmental exposures, socioeconomic status, and psychosocial aspects, making risk determination based on identification of these factors difficult and inaccurate. Yet, defining children at-risk early is utterly critical to improving future mental health outcomes. Due to the unique regulatory features of imprinted genes and their sensitivity to environmental exposures, genomic imprinting has been proposed as an ideal integrated measure of the intrauterine environment for use in epidemiologic studies of the developmental origins of health and disease. As imprinted genes in the placenta can impact the function of this critical organ in directing fetal development and programming, there is also strong evidence to support establishing and validating the link between alterations in imprinting and newborn neurodevelopmental outcomes. Compelled by strong rationale and by emerging evidence, including work from our laboratories linking placenta imprinted gene expression to infant neurodevelopment, this project aims to develop an imprinting-based biomarker that can prospectively predict neurobehavioral outcomes, which ultimately can be used for immediate identification of infants at-risk in order for early intervention to be initiated/implemented. We have developed a multi-stage research plan to first utilize the comprehensive resources of the ongoing Rhode Island Child Health Study (RICHS), which employs the validated and prospectively predictive NICU Network Neurobehavioral Scales (NNNS) as a phenotypic measure of newborn neurobehavior in a birth cohort of 900 newborn infants, to define a biomarker panel associated with key neurobehavioral measures. We will then demonstrate the validity and generalizability of this biomarker using an established but independent resource, the New Hampshire Birth Cohort Study (NHBCS), which uses similar data collection procedures as RICHS. In addition, although the environment is extraordinarily complex, we have decided to focus on fetal exposure to metals, specifically those which are widely considered neurotoxins or those considered protective, as a paradigm to build a comprehensive model to examine the inter-relationships among in utero trace metals exposure, genomic imprinting, and newborn neurobehavioral outcomes. Identification of an imprinting signature associated with abnormal neurodevelopment or environmental exposure would have significant clinical and public health implications by pinpointing certain environmental risk factors for neurobehavioral defects or serve as a basis for early diagnostic tools thus providing an opportunity for early intervention.

Project 1 is central to the overall Children's Environment and Disease Prevention Research Center at Dartmouth. Accumulating data suggest a variety of adverse effects that may result from arsenic exposure during the vulnerable window of fetal development and early childhood. Altered immunity is among the key pathways identified as affected by arsenic in experimental studies and in highly exposed populations. However, such studies do not exist for the U.S. at levels relevant to our population. Infections are the most common causes of illnesses in children in U.S., and are the leading causes of deaths in children worldwide. Additionally, allergy and atopy have become a more widespread source of childhood morbidity in recent years for reasons as yet unknown. Our preliminary data from animal and epidemiologic studies provide both mechanistic and clinical evidence of aberrant immune function related to arsenic exposure in infants. Recent studies also suggest a significant impact of certain environmental exposures on vaccine response in children, but the effects of arsenic on vaccine response have not been investigated. Further, new technologies are just beginning to uncover the fundamental role of the establishment of the intestinal microbiome early in life on immune system maturation and competency as well as in metal biotransformation. We propose to build on a prospective cohort study to determine whether prevalent exposure levels of arsenic influence children's immune response including risk of infection, allergy/atopy, vaccine response and intestinal microbial acquisition. The longitudinal birth cohort study comprises 1,000 mother-child dyads who are residents of New Hampshire and obtain household water from private wells, a potential source of arsenic exposure in the region. We will actively follow infants enrolled in the cohort through: (1) interval interviews with the mothers every four months and (2) screening pediatric records up to age 5 years. We will request a venous blood sample from infants during their one-year well-child visits to test for antibody response to diphtheria/tetanus/pertussis, pneumococcus and polio vaccines in collaboration with Dr. Kari Nadeau at Stanford University. As an exploratory aim, we will sequence bacterial DNA from repeated stool samples (collected at six weeks and 12 months of age) to assess the relation between in utero and early life exposure to arsenic on the developing microbiome. To our knowledge, this is among the first prospective molecular epidemiologic studies of arsenic and children's health in the U.S., and among the few cohorts in the U.S. with detailed early childhood infection information along with other immune-related variables.

Exposure to high doses of arsenic-contaminated drinking water has been shown to have a significant adverse impact on prenatal growth and childhood cognitive development. Much less is known about the child health effects of the lower doses of arsenic exposure typical of U.S. populations despite evidence of adverse effects such as increased cancer risk at such levels in adults. Recent research suggests that diet may be the most significant source of exposure to arsenic for the general U.S. population, particularly children. Understanding the roles of diet and water as sources of arsenic is vital to assessing exposure risk especially in young children for whom developmental toxicant exposure can be highly deleterious. The New Hampshire Birth Cohort Study (NHBCS) recruits from a population where about 15% of households have private wells with arsenic concentrations above the current maximum contaminant level of 10 ug/L. Thus, it provides a unique opportunity to evaluate early childhood arsenic exposure via drinking water and diet and to quantify physical growth and neurodevelopmental effects of such exposure. To characterize early childhood arsenic intake, we will combine consumption data from repeated dietary assessments with arsenic concentrations measured in each child's drinking water and foods. We will then explore how intake through water and diet are associated with urinary and toenail biomarkers of arsenic exposure. These arsenic biomarkers will then be evaluated for their association with altered growth (weight, length/height), and adiposity (body mass index, waist circumference, skinfold thicknesses) during the first five years of life, as well as impairment in behavioral skills and executive function at ages 3 and 5 years, and decrements in cognitive ability and motor proficiency at age 5 years - all neurodevelopment skills for which prior research supports likely arsenic sensitivity. The in utero and early childhood periods are windows of heightened vulnerability to environmental contaminants because of lower body mass and rapid development. Children also have specific dietary patterns that often include foods high in arsenic, especially rice and rice-based products. This innovative project will combine detailed longitudinal assessment of arsenic exposure and growth and neurological development to better understand critical children's health impacts of this common contaminant.

There is strong evidence that the environment experienced in utero contributes to cardiovascular, metabolic, immunologic, and nervous system health during both childhood and adulthood. Defining children at-risk early in life is critical to improving health outcomes; biomarkers of exposure and outcome are powerful tools to use in risk identification and offer an opportunity to inform the mechanisms involved in the developmental origins of disease. Emerging evidence, from our own laboratories and others, suggests that the placenta, as a master regulator of the intrauterine environment and of fetal development, plays a critical role in the developmental origins of health and disease. Thus, the placenta is an ideal tissue in which to identify functional biomarkers that mediate fetal programming of health. In published and preliminary studies we have demonstrated that exposures, such as arsenic, can alter the patterns of DNA methylation and of gene expression in primary human placental tissue, and we have demonstrated that such alterations are associated with fetal growth and newborn neurobehavioral measures. We aim here to develop novel biomarkers of exposure and outcome by examining the hypothesis that environmental exposures encountered by a pregnant woman impact the epigenetic and gene expression profiles of the infant's placenta and these functional alterations are associated with poor-health outcomes in the children. We will employ the existing resources of the ongoing New Hampshire Birth Cohort Study, and its established biorepository of placental tissues, to allow sensitive and robust assessment of gene expression and genome wide DNA methylation in combination with infant and maternal biomarkers of arsenic exposure. We propose to use state-of-the-art genomic technologies to identify and validate novel profiles of DNA methylation and gene expression susceptible to environmental exposure in human placenta and to examine their association with newborn outcomes including growth, infection and neurodevelopment being assessed in Projects 1 and 2 of the Program. These examinations will provide critical insight into the mechanisms of the developmental origins of lifelong health, highlight novel pathways affected by exposures that drive children's health, and identify novel biomarkers which can be used to find at the earliest points of life, children at-risk so necessary interventions can be employed soon enough to prevent future disease.

Arsenic (As) is a common environmental toxicant to which millions of people in the U.S. are exposed via contaminated drinking water. Arsenic has been linked to increased risk of infectious disease and, possibly, other manifestations of immune dysfunction but there is uncertainty about the mechanism and full scope of its potential immunotoxicities. Emerging evidence suggests that As may perturb T cell function, including both effector T cells and T regulatory cells (Treg), a subset of T cells identified by our laboratory and others to play a critical role in suppressing inflammation. Moreover, many immunotoxicants have profound effects when exposure occurs during prenatal development of the immune system. Yet, there are few, if any, studies of the relation of prenatal As exposure with specific measures of immune function. We propose to assess the relation of in utero As exposure (measured via maternal pregnancy urine As) with immune function at birth and age 1 year. The study will build on prospectively collected exposure, biospecimen, and extensive covariate and health data available from an ongoing cohort study of pregnant women and their infants in New Hampshire. Our hypothesis is that there will be a link between in utero As exposure and immune dysfunction, including impaired T cell and Treg function (in cord blood and placenta), which can lead to decreased responses to vaccinations, and possibly, increased infant susceptibility to infection and atopy. To test these hypotheses we will determine whether in utero As exposure is associated with: 1) impaired T cell and Treg function in infant cord blood samples, and 2) decreased immune indicators of placental tolerance. Also, we will 3) conduct exploratory analyses of T cell and Treg function in umbilical cord blood as predictors of enhanced risk of infant infections and atopic disorders as assessed during the 1st year of life, and 4) test the feasibility of determining T cell function, vaccine response and IgE levels in 1 year olds and their relation to As. We have established a new interdisciplinary collaboration between a clinical-scientist investigator with expertise in immunity and environmental and perinatal epidemiologists to advance understanding of the relation of a common environmental contaminant, As, with alterations in immune function and related childhood diseases. If the aims of our study are met, the results will be the basis for a more in depth study to confirm and extend our work and to develop new strategies for biomonitoring of immune function. Moreover, our study can be used to identify remediable environmental risk factors for prevalent childhood conditions with substantial public health importance, including infectious and atopic diseases.

Both animal and in vitro studies support an association of low level arsenic exposure with impaired immune function as reflected in suppression of innate immunity and increased pathogen load. The few epidemiologic studies of this association are primarily from heavily exposed populations, and often involve small sample sizes or rely on ecologic exposure measures, and may not be generalizable to other regions of the world. Arsenic exposure in these heavily exposed populations has been variously associated with an increased risk of respiratory infection, bronchiectasis, and parasitic infection as well as with changed markers of immune function including, e.g., cytokine levels (IL-7, IL-2) and T-cell (CD4/CD8) ratios. The overall goal of this study is to assess the relationship of environmentally relevant levels of arsenic with maternal and infant immune function among 1,000 women and infants enrolled in an ongoing pregnancy cohort study of reproductive toxicities of arsenic. This ongoing longitudinal study is being conducted among mother-infant pairs who are residents of New Hampshire and obtain household water from wells which are a potential source of arsenic exposure. Specifically, we will expand this ongoing study to test the following new hypotheses: (1) prenatal and early life arsenic exposure (via water, food) is associated with an increased risk of infection during the 1st year of life; (2) arsenic exposure is associated with an increased risk of infection during pregnancy. Secondarily, we will assess the relation of pre- and post-natal arsenic exposure with vaccine response at age one year (antibody titers to tetanus and diphtheria) and whether individual variation in arsenic metabolism (maternal urinary metabolite profiles and polymorphisms in arsenic metabolism genes) and other factors (e.g., smoking, folate intake) modify the effects of arsenic on infant or maternal infection. Altered immune function, particularly in pregnancy and early childhood, can have a profound impact on both perinatal and subsequent health. To the best of our knowledge, studying immune effects of arsenic in a U.S. population of mothers and infants with both individual biomarkers of exposure and measures related to likely susceptibility, has not been done previously.

Exposure to metals during development and early in life is a critical public health issue, and both water and food can be vectors of exposure, especially to inorganic arsenic. However, limited data are available on the dietary exposure to metals for U.S. infants or the subsequent expression of this exposure in established biomarkers. Recent data, including our own preliminary results, suggest that rice and rice products are significant contributors to inorganic arsenic exposure. Building on the Dartmouth Superfund Program's prospective pregnancy cohort, this pilot project will explore dietary sources of metal exposure in a cohort of infants through the first year of life. The goal is to identify the contribution of diet to both metal exposure and the ability to reduce metal toxicity via potential modifying factors such as folate, iron, and vitamin B. The project will focus particularly on exposure through breast milk and infant formulas, but will also collect data on the period when infants transition to solid foods, especially rice cereals. Information regarding the diet of the infant (including breastfeeding; amounts of formula, cereals, solids, and water being consumed; and vitamin supplementation) will be collected every 2-3 months during the first year of life via a phone questionnaire. Toenail clippings and a urine sample will be taken from the infant at ~4 months, and lactating women who are using a breast pump to store milk for their infants will be encouraged to provide a sample of their milk at the same time. We will also obtain toenail clippings from the infant and mother at ~12 months, together with information about maternal diet over the past year using a food frequency questionnaire. We will compare the infant dietary data with arsenic in the toenail and urine samples, and also use data on arsenic metabolites in the urine sample as a marker of infant ability to metabolize arsenic postpartum. If we are able to identify the sources of infant metal exposure, or factors that appear to mitigate the effects of this exposure, we can initiate intervention studies to identify specific dietary recommendations for prevention of adverse outcomes.

Arsenic readily crosses the placenta and is teratogenic in animal models. However, few epidemiologic studies exist on the potential teratogenic effects of arsenic in humans. Scant prior data from the U.S. and emerging data from Bangladesh suggest a link between drinking water arsenic and occurrence of congenital anomalies. We propose to conduct a pilot study integrating geospatial and epidemiological data to characterize the spatial distribution of birth defect occurrence in New England in relation to environmental exposures, in particular inorganic arsenic. In this pilot study: 1) We will quantitatively characterize the spatial distribution of birth defect occurrence and determine the presence of any high-risk areas (i.e., birth defect hot spots); 2) We will quantitatively evaluate spatial associations between birth defect occurrence and water arsenic concentrations; 3) We will conduct a pilot case-control study (as a feasibility study) to investigate at the individual level the associations between birth defect occurrence and arsenic and other environmental exposures. Geocoded data on birth defect occurrences will be made available through the New Hampshire Birth Conditions Program. We will estimate drinking water arsenic exposure using public databases combined with a large data set of private water systems collected through epidemiologic studies in the state in collaboration with the USGS. We will test whether an excess in major and minor birth defects are associated with drinking water arsenic exposure using geographical information systems (GIS) and a specialized software package we designed for a previous study of radon and lung cancer to be extended as part of this pilot study. We will further assess the feasibility of conducting a case-control study of birth defects in the region (i.e., with the collection of individual biomarker data on arsenic), and conduct preliminary analyses of the arsenic-birth defects associations on an individual level. To our knowledge this will be the first attempt of this kind to investigate arsenic exposure and congenital anomalies in a U.S. population.

Project 4 is an integral component of the Dartmouth Superfund Research Program. Over the past 18 years, we designed and tested methods of measuring environmentally relevant levels of exposure to metals and applied novel biomarkers of exposure, susceptibility, and early response to large- scaled, population-based epidemiologic studies in the US. To date, we have tested over 8,000 households for arsenic (As), of which over 3,500 had private water systems. A GIS analysis of the data (performed in collaboration with the Trace Element Analysis Core) revealed distinct "clusters" of high household water As levels. Since 2009, we successfully established a pregnancy cohort of women who use private wells in one of these cluster regions. This initial funding period was designed to examine the relationship between in utero exposure to arsenic using multiple measures of exposure (drinking water, toenails and urine) on birth outcomes (e.g., fetal growth and gestational age). We also sought to test the hypothesis that methylmercury intake alone, or in combination with other factors, influences fetal growth and gestational age. We plan to conduct a collaborative analysis with the NIEHS-funded New Bedford birth cohort study (adjacent to a Superfund Site). Of the household tap water samples tested thus far, ~15% exceeded the maximum contaminant level for As established by the US EPA of 10 ug/L. While evidence suggests that As is related to adult onset diabetes and hypertension, its effects on these outcomes during pregnancy are uncertain. Thus, we will determine whether As influences glucose and blood pressure control during pregnancy and identify potential genetic susceptibility loci for these effects. Additionally, we will test pregnant women and newborns for markers of systemic inflammation and vascular endothelial dysfunction that have been previously found to relate to As exposure among adults in more highly exposed regions. Our hypothesis is that pregnancy and fetal development represent "windows" of susceptibility to the effects of As on cardiometabolic outcomes. To our knowledge, our study is one of the only molecular epidemiologic investigations of pregnancy and early life exposure to As in vulnerable subgroups of the general population of the U.S. Our goal is to inform risk assessment and management of toxic metal exposure in the U.S., and aid early intervention strategies to prevent adverse health effects from these exposures.