Although it is well known that humans are exposed to a complex mixture of different chemicals, having constit- uents that change dynamically as an individual ages, very little is known about how these exposures interact to impact health outcomes. The overarching focus in the toxicology and epidemiology literatures has been on ex- amining the health effects of chemicals one at a time. One reason for the lack of consideration of more holistic approaches for simultaneously assessing the health effect of multiple chemicals is the lack of appropriate sta- tistical methods that are interpretable and reliable at disentangling the impact of each chemical in the mixture. When attempts are made to include different chemicals simultaneously in statistical models, most of the focus has been on generic multivariate statistical methods that often fail to have adequate performance. For exam- ple, simply including different exposures in nonparametric regression models can lead to unstable estimates due to the so-called curse of dimensionality, particularly if the different exposures are moderately to highly cor- related. The overarching goal of this proposal is to develop novel statistical approaches, which are specifically tailored for mixture exposure problems, incorporating mechanistic constraints and supplemental data on chem- ical structure and toxicological responses to improve performance. An initial focus is on developing restricted nonparametric regression methods, which constrain the response surface to be monotone with possible down- turns at low and high doses, consistent with prior data and mechanistic knowledge. Such constraints substan- tially improve stability and performance in estimating dose response, while facilitating interpretation. Another key advance is the development of mechanistic interaction models, which reduce dimensionality and enable disentangling of main effects and chemical-chemical interactions, allowing no interaction, synergy or antago- nism. A further thread designs a novel class of mechanistic response surface models, which directly incorpo- rate supplemental data on chemical structure and borrow information from one-chemical-at-a-time toxicological studies. These models enable de novo prediction of dose response and interactions for new chemicals, which have known structure but have not been studied in toxicology and epidemiology studies. These predictions in- clude an accurate characterization of uncertainty, highlighting cases in which more data are needed. To be ap- propriate for a rich variety of epidemiological study designs, the methods are generalized to account for covari- ate adjustments, longitudinal and nested data structures, censoring, and other complications. A key focus of the project is on producing user-friendly software that non-statistician scientists can use to analyze and visual- ize the health effects of mixture exposures, provided on the project's GitHub site and beta tested. Methods will be tested in a multi-tiered fashion through theoretical studies, comprehensive simulation experiments including comparisons to a rich variety of existing approaches under challenging scenarios, and applications to multiple epidemiology studies. These studies include the MSSM Children's Cohort, NHANES, and CHAMACOS.

Since its inception in 1999, the CHAMACOS study is one of the longest running cohort studies examining the impact of early life environmental exposures on neurodevelopment, growth, and respiratory disease and the only one focused on low-income, Latino children in a farmworker population. We have collected extensive health, exposure, demographic, neighborhood, and regional data, as well as biological (e.g. blood, urine, breastmilk, hair, saliva, deciduous teeth) and environmental (e.g. dust, allergens) samples at multiple visits and have created a large biorepository with more than 220,000 samples stored for future use. With over 140 publications, CHAMACOS is a successful and well-established environmental epidemiology cohort. We have used banked specimens and archived data to demonstrate relationships of pre- and postnatal exposures to pesticides, flame retardants, and other chemicals with poorer neurodevelopment, reduced lung function, obesity, and other outcomes. We have shown that environmental exposures affect a multitude of molecular mechanisms that influence health, such as PON1 enzymatic activity, adipokine and isoprostane levels, DNA methylation and miRNA expression. The CHAMACOS resources have supported multiple NIH, EPA, and non-federal grants and trainees, including collaborations with other institutions. However, the infrastructure required for management of this vast trove of data, the laboratory facilities to ensure the safety of hundreds of thousands of samples, and the effort to keep families engaged and participating in this long- running study have increased over time, while funding for these activities has decreased. The aims for this proposal are to (1) retain participation in the cohort through community engagement, (2) maintain and strengthen data management infrastructure, (3) maintain and enhance the existing biorepository through replacement, repair, and maintenance of aging deep freezers to ensure the integrity of samples and improvements to systems to track samples used for multiple research grants, pilot studies, and collaborations, (4) conduct validation, pilot, and feasibility studies to investigate new methods of assessing environmental exposures, develop best practices for biorepositories, and explore novel methodologies related to metabolomics, genomics, and the microbiome, and (5) develop a data sharing portal to encourage use by outside collaborators of existing CHAMACOS data. In summary, infrastructure support for the CHAMACOS cohort study will preserve specimens, ensure well- documented data for future studies and data sharing, and maintain participant retention, assuring effective future use of these valuable resources. This maintenance grant will thereby strengthen our ability to answer key questions about the impact of environmental exposures on health over the life course and will assure that the extensive resources generated by the CHAMACOS study will be effectively used by investigators worldwide for years to come.

The alteration of gene expression mediated by epigenetic modifications, specifically DNA methylation, has been proposed as a mechanism by which chemical and biological factors during gestation and childhood may influence health and adult disease onset. Epigenetics of imprinted genes, which exhibit expression of one parental allele, represent a promising area of fetal programming research, as many imprinted genes are involved in early growth and development. The aim of the proposed study is to characterize the relationship of prenatal exposure to common endocrine disruptors, phthalates, with DNA methylation of imprinted genes in newborns. The analysis will include DNA samples from the well-characterized CHAMACOS birth cohort study of hundreds of Mexican-American children and their mothers followed from early pregnancy through adolescence. In order to discern relationships between epigenetic profiles of newborns and early life exposure parameters, DNA methylation of imprinted genes in newborns will be assessed using targeted pyrosequencing, followed by validation of the hits by next generation sequencing. DNA methylation of imprinted genes will also be interrogated for associations with biological factors (sex, gestational age, and birth weight), phthalate exposure during pregnancy, and parental obesity. The CHAMACOS study provides a unique opportunity to assess biological and environmental interactions with imprinted gene epigenetics in a cohort with substantial environmental stressors and a high prevalence of parental pre-pregnancy obesity. The research outlined in this proposal will contribute to the limited knowledge regarding the effects of in utero phthalate exposure on methylation of imprinted genes, which thus far includes limited data on only two imprinted genes.7,8 We will expand this analysis to include several additional imprinted genes with high biological significance for fetal development. We will also validate findings of the association between methylation marks and parental obesity from another birth cohort (NEST).9,10 Data generated by the state-of-the art epigenetic and bioinformatic methodologies will provide insights into imprinted gene epigenetics and may identify potential new targets for intervention in pregnant women.

For the past 15 years, we have chronicled the development of over 600 primarily low-income, first- generation Latino children born in the Salinas Valley, known as the nation's salad bowl. The children of the CHAMACOS longitudinal birth cohort are now coming of age in a community identified by the Department of Justice as an epicenter of youth gang violence. Our previous research has shown that in utero exposure to organophosphate pesticides (OPs) was associated with impaired attention3 and lower IQ at school age for CHAMACOS children,4 both of which are risk factors for adverse behavioral outcomes in adolescence and adulthood. Our data also indicate that CHAMACOS children have experienced considerable early life stressors, including poverty, food insecurity, housing instability, household overcrowding, family conflict and separation, maternal depression, and fear of deportation that may predispose them to adverse outcomes. We propose to investigate the interaction of in utero exposure to a host of neurotoxic pesticides with early life social adversity in association with behavioral outcomes during the transition from adolescence to early adulthood (age 16 and ~18 years): specifically, externalizing and risk-taking behaviors, delinquent and criminal activities, and school failure versus success/graduation. Through our research, we will investigate developmental outcomes of exposure to the complex mixture of pesticides used in Salinas Valley agriculture, making use of geo-coded that allows assessment of chemicals that lack biomarkers. We will also collaborate with child development Pesticide Use Reporting data (PUR) experts to synthesize the wealth of data we have gathered on family-level and neighborhood-level adversities (e.g. poverty, crime) into cumulative adversity exposure variables corresponding to specific developmental windows (i.e. in utero, birth to age 5, and birth to age 9), and will do the same for protective factors (e.g. maintenance of positive cultural values, child-parent attachment). We hypothesize that exposure to neurotoxic pesticides and early life adversity will each independently increase adverse behavioral outcomes in adolescence/early adulthood, and that early adversity may modify the effects of pesticide exposures. We further suggest that the decreased cognitive abilities and poorer attention observed in association with pesticide exposure in CHAMACOS children at school age may mediate this relationship. Thus, the goal of this project is to evaluate the neurotoxicity of current-use pesticides and early social adversity to human populations, assess effects of early life exposure to both these chemical and non-chemical stressors on adolescent/early adult behaviors of societal concern, and identify targets for early intervention to prevent longer term poor outcomes.

Phthalates are synthetic chemicals used in personal care products and in flexible plastic products. Along with their metabolites, phthalates are rapidly excreted in humans with half-lives of 5-24 hours. Exposure to phthalates is ubiquitous because of their widespread use and their ability to leach easily into the environment. Phthalates are endocrine-disrupting chemicals that may interfere with neuroendocrine pathways involved in neurodevelopment, including anti-androgenic activity and thyroid hormone homeostasis. The prenatal period is a particularly sensitive target for phthalate exposure due to rapid brain development. Previous studies of phthalates and neurodevelopment are limited by small sample size, cross-sectional design, and limited data on neurodevelopment. The primary objective of the proposed research is to investigate the association of prenatal exposure to phthalates with cognitive and behavioral development in childhood and adolescence. We will examine these relationships in the Center for the Health Assessment of Mothers and Children of Salinas (CHAMACOS), a large, well-characterized longitudinal pre-birth cohort of mothers recruited 1999-2000 during pregnancy and their offspring. We will analyze existing data on maternal phthalate levels, measured twice during pregnancy (~13 and 26 weeks), in relation to the cohort's extensive data on neurodevelopment measured from age 7 years until adolescence for 336 children with exposure and outcome data. We will examine associations with the following outcomes: (1) general cognition, (2) executive function, (3) attention problems, externalizing behaviors and internalizing behaviors, and (4) social cognition. This study will take advantage of the CHAMACOS cohort's longitudinal data collected on teacher- and parent-rated scales as well as objective, performance-based tests. Additionally, this will be the first study to examine associations of phthalates with neurodevelopmental measures in adolescence, a critical time for manifestations of early life exposure effects. Results of this study, which will further our understanding of the long-term impact of early-life exposure to phthalates on neurodevelopment, have important policy implications given that widespread exposure to these chemicals in personal care products is modifiable and alternatives may be available during sensitive time windows, such as pregnancy.

Exposure to triclosan, an antibacterial agent used in soaps, toothpaste, and other personal care products, is widespread, with 75% of Americans having detectable levels in their bodies. In vitro and animal studies show that triclosan is an endocrine disruptor. In rodent studies, triclosan exposure during critical windows of development lowers levels of the thyroid hormone thyroxine (T4), increases allergic sensitivity and asthma-like response, decreases testosterone in males, and alters pubertal timing in females. To date, only a small number of studies have examined health effects of triclosan in humans, focusing on school-age children and adults. These studies provide some confirmation of effects on endocrine and immune systems, but they do not address exposure in utero - a critical period for endocrine disruption and immune development - and thus likely underestimate triclosan's potential health impact. Both the U.S. Food and Drug Administration (FDA) and Environmental Protection Agency (EPA) are embarking on comprehensive regulatory reviews of triclosan, but are challenged by a dearth of human data. High-quality, independent research assessing associations of prenatal and early childhood triclosan exposures with subsequent health effects are urgently needed. The Center for the Health Assessment of Mothers and Children of Salinas (CHAMACOS) is a longitudinal birth cohort study that has gathered comprehensive data on environmental exposures, health, and development in more than 300 children who have participated from before birth through age 12. We propose to characterize urinary triclosan concentrations from CHAMACOS mothers during pregnancy and their children at 5 and 9 years of age and to examine the potential impact of this exposure on: 1) thyroid hormone homeostasis in pregnant women and newborns, 2) allergy and asthma symptoms in children at ages 5 and 7, and 3) timing of puberty and sex hormone levels in children between ages 9 and 12. We have already collected most of the data needed to achieve these aims, and crude, preliminary analyses suggest associations of triclosan with decreased T4 and increased risk of asthma symptoms in this cohort. However, the work needed to finalize urinary triclosan measurements, obtain additional outcome data, and conduct sophisticated data analyses that control for longitudinal effects and confounding cannot be completed without dedicated funding. This study is exploratory, because it is the first longitudinal study of developmental triclosan exposure; developmental, in that it lays groundwork for future in-depth research; and high risk, because we hope to have results in time to inform pending policy decisions. By relying on a rich existing data, this project can be completed in just two years, promising high regulatory and scientific impact.

Neurobehavior and cognition in children are affected by hereditary, environmental and social factors, but the molecular mechanisms remain largely unknown, prompting the study of the potential role of epigenetic dysregulation. Epigenetic mechanisms control gene expression without changes in DNA sequence, and DNA methylation and non-coding RNAs are two important types of epigenetic modifications. Recent studies have demonstrated associations between the multifunctional antioxidant enzyme paraoxonase (PON1) and poor neurodevelopment and adverse health outcomes in children. We previously characterized PON1 functional genomics in Mexican-American children and their mothers from the CHAMACOS study, and found a greater than 100-fold range of PON1 enzyme activities defined in part by age and genotype. Although genetics influence 25% of PON1 expression, a large portion of PON1 variability remains unexplained suggesting a role for other factors such as epigenetics. In fact, some studies have shown that epigenetic dysregulation can affect neurodevelopment and cognitive processes such as learning, memory, and neurogenesis. Among CHAMACOS children, PON1 genotype and enzyme levels were associated with changes in behavior and learning at age 2 and lower IQ scores (measure of cognitive abilities) at age 7. While no studies on the role of PON1 epigenetics on gene expression and poor cognition have been reported, our preliminary data show that DNA methylation in PON1 is strongly associated with PON1 enzyme levels. We hypothesize that epigenetic mechanisms significantly contribute to the broad variability in PON1 enzyme levels and poor neurodevelopment in children. The proposed study will take advantage of the well characterized data on PON1 genetics, rich collection of biological samples and neurobehavioral data already amassed through the CHAMACOS study. Our specific aims are to (1) determine the relationship between maternal PON1 DNA methylation profiles during pregnancy with those of their children at birth and age 7; (2) characterize expression of miRNAs that bind to PON1; (3) examine the relationship between epigenetic marks and PON1 expression; and (4) establish the effect of PON1 epigenetics on attention, motor function, and cognition in 7 year olds. The total sample size will be 315 mother-child pairs with complete neurodevelopment assessment, PON1 genetic and enzyme level data, and biological samples for all proposed analyses. This work will serve as model for studies of candidate susceptibility genes and will address several key knowledge gaps in environmental molecular epidemiology. Understanding how epigenetic mechanisms affect child neurodevelopment and PON1 variability in pregnant women and children will help to identify vulnerable sub populations. Environmental exposures can result in epigenetic alterations, thus characterization of the relationship between epigenetics and disease will establish the foundation necessary for examining mediation of gene environment interactions by epigenetics.

In a risk assessment of air monitoring data by the California Health Department, the top four pesticides ranked by non-cancer health risk, based on respiratory and neurological toxicity, were metam sodium, methyl bromide (MeBr), 1,3-dichloropropene (1,3-DCP) and chloropicrin. These fumigants, which are used to sterilize soils prior to planting many crops, account for ~20% of the 78 million kilograms (kg) of pesticides used annually in California. In the U.S., approximately 50 million kg of fumigants are applied each year, primarily in California, Florida, Washington, Idaho and Oregon, representing about half of worldwide fumigant use. Soil fumigants are a concern for nearby residents due to their toxicity and potential for drift. All four of these fumigants are known respiratory toxicants, and MeBr, chloropicrin and metam sodium are also neurotoxic and genotoxic. Due to concerns about the Earth's ozone layer, MeBr was banned by the Montreal Protocol in 2005. However, under an EPA exemption, MeBr is still used in large quantities in California. Since 2005, use of replacement fumigants, including chloropicrin, 1,3-dichloropropene (1,3-DCP) and metam sodium has increased by 20% in the state, and this trend is expected to continue. There are no biomarkers available to assess non-occupational exposures to fumigants. Thus, geospatial methods must be used to estimate exposure from nearby applications. The California Pesticide Use Reporting (PUR) system, considered the best in the world, requires reporting of all agricultural pesticide use. For this proposal, we will use neurodevelopmental assessment and respiratory health data from the CHAMACOS cohort to determine the association of residential proximity to MeBr, chloropicrin, 1,3-DCP and metam sodium applications during pregnancy and early childhood with cognition, motor functioning, attention and pulmonary function in 7-year-old children. Further, we will confirm our findings of an association between prenatal MeBr exposure and adverse birth outcomes (birth weight and gestation length) with statewide geocoded PUR information and vital statistics data (n~750,000). This will be the first study to: 1) Examine the relationship between in utero and early childhood fumigant exposure and neurodevelopment and respiratory health in school-age children; and 2) Utilize statewide fumigant use and vital records information to determine the association between prenatal residential proximity to fumigant applications and birth outcomes in a large population. Virtually nothing is known about pregnant women's and children's exposures to these chemicals or their long-term health effects in children. This research will generate new information about the health effects of fumigants in school-age children that will inform the design of future epidemiological and risk assessment studies as well as on-going policy decisions regarding the replacement of MeBr and the implementation of new strategies to reduce fumigant exposure in agricultural communities.

In the last 30 years, the prevalence of obesity has risen sharply among children, and minority populations are particularly vulnerable. In the CHAMACOS birth cohort, a Mexican-American population from the agricultural Salinas Valley, CA, children have a particularly high prevalence of obesity (>39% at age 9) compared to both Mexican-American children (25%) and children of all races (20%) in the United States. Changes in diet and physical activity patterns do not completely explain the current obesity epidemic. Recent research suggests that exposure to environmental obesogens, particularly during the prenatal period, may also contribute to increases in obesity. Evidence from animals and epidemiologic studies suggests that phthalates, components of plastics and cosmetics ubiquitously found in humans, may be obesogenic. For instance, as shown in animals, they can change levels of biomarkers of obesity such as adipokines, protein hormones that regulate adipogenesis. There is also growing evidence that environmental exposures can influence DNA methylation (one of the main types of epigenetic markers) and result in adverse health outcomes. However, to date, no data are available on the effects of prenatal phthalate exposure on the molecular mechanisms of obesity in children or the potential role of epigenetic dysregulation. The overall goal of this proposal is to determine whether prenatal phthalate exposure contributes to the development of childhood obesity and metabolic syndrome (MetS) and to examine whether epigenetic changes mediate this relationship. We hypothesize that in utero phthalate exposure alters DNA methylation and leads to changes in adipokines (adiponectin and leptin), oxidative stress (isoprostanes), and higher risk of obesity and metabolic MetS in children. First, we will assess whether early and/or late pregnancy exposures are associated with a) biomarkers (adiponectin, leptin and isoprostane) and b) parameters of obesity and MetS in children at 5, 9 and 14 years of age. Second, we will characterize DNA methylation patterns in children at birth and 9 years of age, and examine whether site-specific changes in the methylome (by 450K array) are associated with a) prenatal phthalate exposure and b) biomarkers and parameters of obesity and MetS. Finally, we will validate 450K array data using next generation bisulfite sequencing and the most advanced expression and bioinformatic methodologies. The proposed research utilizes the rich collection of biological samples and data on exposures, diet, activities, and health outcomes in CHAMACOS participants. By extending assessment of children to age 14, this study will include critical periods of obesity development spanning pregnancy through adolescence. Our results will help elucidate the longitudinal effects of phthalates on obesity, explore the role of epigenetics and adipokines in the development of obesity and MetS, and address important gaps in our understanding of obesity. They will also inform policies aimed at improving children's health and preventing child obesity.

Organophosphate flame retardants (OPFRs) persist in the environment and have been used in consumer products since the 1970s. Restrictions on the use of penta-polybrominated diphenyl ether (PBDE) flame retardants have resulted in increased use of OPFRs as replacements, including the OP triesters [tris(1,3- dichloro-2-propyl) phosphate (TDCPP), tris(chloropropyl) phosphate (TCPP), tris(2-chloroethyl) phosphate (TCEP), and triphenyl phosphate (TPP)]. These chemicals are applied to polyurethane foams used in furniture, child car seats, and related products to meet federal and state flammability standards. OPFRs have been measured in >95% of house dust samples collected in the United States, as well as in the foam of numerous baby products. In another recent study, these compounds were detected in >95% of urine samples from adults. Animal research suggests that OPFRs may affect neurodevelopment through non- cholinergic mechanisms similar to some organophosphate (OP) pesticides. Despite the widespread presence of these compounds in home environments, and their structural similarity to neurotoxic OP pesticides, understanding of human exposure and health effects of OP triesters is scarce. To date, no studies have examined OPFR exposures and health effects in vulnerable populations, such as pregnant women and children living in California, where stricter flammability standards have resulted in very high flame retardant exposures. Recently, we developed laboratory methods to measure metabolites of the high-use OPFR flame retardants TDCPP and TPP in urine. For this proposal, we will use specimens and data from the CHAMACOS study, an ongoing NIEHS-EPA funded birth cohort study of low-income residents in the Salinas Valley, CA. We propose to measure OPFR metabolites in mothers' urine and examine the association of prenatal OPFR exposure and neurodevelopment in their children at 7 years (n=330). We will also measure OPFR levels in dust samples collected from a subset of these CHAMACOS women's homes (n=125), determine the correlation between OPFRs in urine and dust, and identify predictors of home OPFR contamination. This will be the first study to: 1) evaluate predictors of OPFR contamination in homes; 2) report levels of biological measurements of exposure to OPFRs in pregnant women; and 3) study the relationship between in utero OPFR exposure and human neurodevelopment. This new information about human exposure to OPFRs will support the development of more comprehensive studies by applying innovative scientific methods to address key knowledge gaps about OPFR exposure and health effects.

Oral diseases are amongst the most common chronic diseases in the US and result in significant disability in the most vulnerable sections of the population, especially children and the poor. The effects of dental diseases extend beyond the oral cavity, with evidence linking poor oral health to diminished quality of life and systemic disorders including cardiovascular disease and cancer. It is, therefore, important that the environmental determinants of oral diseases are systematically investigated to identify their risk factors. Furthermore, metals are deposited in teeth and can be used as biomarkers of fetal/childhood exposure. The use of laser ablation to tease out concentrations within dental layers that correspond to fetal vs childhood development would be a major innovation as it would allow for reconstructing fetal exposure as opposed to cumulative exposure. One of the major barriers to this field of research is the lack of dentists who specialize in environmental health. Dr. Arora is among the few dentists who have previously undertaken studies on the oral health effects of metal toxicants. For this award, during the K/Mentored phase, Dr. Arora will receive structured training in environmental epidemiology, toxicology, genetics, biostatistics and laboratory methods, and he will also lay the foundation for research to be undertaken in the latter stages of this award. In the R/Independent phase of this award, Dr. Arora will study prospectively the oral health effects of metal exposure in a well-established cohort of mother-child pairs residing proximal to a large Superfund site contaminated with metals in Tar Creek, Oklahoma. He will also collect shed deciduous teeth from children in this cohort as well as two others - the CHAMACOS cohort, Salinas Valley, CA (University of Berkeley), and Mexico cohort, Mexico City (Harvard School of Public Health). He will address two Specific Aims: i) to investigate the prospective relationship of environmental metal exposure with dental caries in children, and the association between metals and dental caries/periodontal disease in adults; and ii) to validate the use of tooth-metal concentrations as a biomarker of exposure to metal toxicants capable of reconstructing prenatal vs early childhood exposure, a major advancement in exposure biology. The PI, Dr. Arora, is a dentist and is well-positioned to carry out the proposed training program and research studies because of his prior training in oral biology and the unique resources available through his mentorship team including access to a large established prospective birth cohort that will provide the necessary exposure assessment data, genetic material, newborn measurements and covariate data during this award period; access to state-of-the-art molecular biological laboratories; and advanced coursework in environmental epidemiology and genetics. The proposed training and career development will enable Dr. Arora to become an independent investigator and position him to play a leadership role in the field of environmental dentistry.

Bisphenol A (BPA) is one of the highest production chemicals in the world. It is used in polycarbonate beverage containers, food packaging, linings of food cans, and sealants for teeth. Public health concerns have been raised about BPA because recent studies demonstrate: 1) that it is an endocrine disruptor that acts as an estrogen at low doses; 2) that it leaches from consumer products into food and water; 3) and that human exposure is nearly ubiquitous. Animal studies suggest that BPA alters sexual and reproductive development, including sexually-dimorphic alterations in brain structure and behavior; increased weight gain; and earlier puberty. Because of these health concerns, several countries and U.S. states are considering legislation to ban BPA. However, almost no studies to date have examined the health effects of BPA in humans. This proposal seeks to address this data gap by examining the health effects of developmental BPA exposure in a cohort of children living in California. The CHAMACOS Study is a longitudinal birth cohort study that has followed ~500 low-income children living in a predominantly Mexican-American, farmworker community from before birth to age 9. Children's growth, health, neurodevelopment, and behavior have been assessed at birth and age 6 months, 1, 2, 3 1/2, 5, and 7 years of age and urine and/or blood samples were collected at each time point. In this rich dataset, the intent is to examine the association of BPA exposure in utero and in childhood with child growth and development. The specific aims are: 1) to measure BPA concentrations in urine collected from ~400 mothers during pregnancy and from their children at 6 months, 5 years, and 9 years of age. All samples, except those at age 9, have already been collected: 2) to determine whether maternal BPA concentrations are associated with maternal (TSH, T3, T4) and child (TSH) thyroid hormone levels. 3) to determine whether maternal and childhood BPA concentrations are associated with cognitive functioning and behavior at age 5 years.; 4) To determine whether maternal and childhood BPA exposure is associated with body mass at age 5 or age 9 years; and 5) To determine whether maternal and childhood BPA exposure is associated with metabolic syndrome at age 9 years. Data has already been collected on thyroid levels in mother and child and neurobehavioral development and body mass at age 5 years. Metabolic syndrome diagnosis will be determined from new data gathered at age 9 (blood pressure, waist circumference, lipids and glucose in fasting blood). This project is uniquely suited for this Grand Opportunities RFA because it uses an extant dataset and banked samples, it focuses on developmental exposures, and it can be begun immediately and completed in 2 years. This is a potentially high-impact project that can provide immediate answers about the human health effects of BPA exposure to legislators and policy makers and lays the groundwork for future investigations. PUBLIC HEALTH RELEVANCE: The proposed research will provide some of the first data on the human health effects of BPA exposure and address key data gaps identified by advisory and regulatory bodies. By relying mainly on existing data and banked samples, we will accelerate the science by completing the research in just two years. In addition, measurement of BPA in this cohort will lay the foundation for future examination of multiple other outcomes, such as asthma (including spirometry), immune function (Th1 and Th2 cytokines), and pubertal onset, in this rich and unique dataset.

Children of farmworkers in California are exposed to a unique mix of chemicals that may affect both their reproductive and neurobehavioral development. Potential exposures include the pesticide dichlorodiphenyltrichloroethane (DDT), which was recently used in Mexico where most of their farmworker parents originated; manganese (Mn), which is a key component of some widely used agricultural fungicides; and polybrominated diphenyl ether (PBDE) flame retardants, which are prevalent in California due to the state's strict flammability standards. DDT, Mn, and PBDEs are endocrine disruptors and neurotoxicants in animals, but studies in humans are limited. Age at onset of puberty in girls has declined in recent decades, possibly due to endocrine-disrupting compounds like DDT, Mn, and PBDEs, but almost no studies have examined pubertal onset in boys. These pollutants may also jeopardize children's cognitive development, particularly with exposure during the prenatal and peri-adolescent periods of peak brain growth. We propose to investigate the associations of DDT, Mn, and PBDE exposure in utero and at age 9 with puberty onset and neurodevelopment in the CHAMACOS longitudinal cohort of Mexican-American boys in the Salinas Valley, California. Boys enrolled in the CHAMACOS Study have been followed since before birth until 7 years of age. We will continue to follow these boys from age 9 to 13 years and enroll ~150 new 9 year-old-boys from the same source population, expanding the study from ~150 boys to 300. We have measured DDT and PBDE levels in prenatal maternal blood (original cohort) and will back-extrapolate prenatal levels from maternal blood collected at 9 years (new enrollees). Prenatal Mn exposure will be measured in boys' teeth. DDT, Mn, and PBDE concentrations at age 9 will be measured in boys' blood. We will assess the relationship of in utero (measured or extrapolated) and age 9 (measured) concentrations of DDT, Mn, and PBDEs with neurodevelopment (specifically general cognition, attention, memory, motor, executive function, and social cognition) and with the timing and tempo of their pubertal onset as assessed by clinical Tanner exams conducted every 9 months between age 9 and 13 years and hormone levels measured at 12 years. Thus, this proposal takes advantage of a rich dataset of Mexican-American children participating in the CHAMACOS longitudinal cohort study and proposes to expand this unique cohort and follow the children into early adolescence.

Persistent organic pollutants (POPs) are lipophilic chemicals that persist in the environment, accumulate in the food chain and human Tissues, and are likely endocrine disruptors. In 2001, more than 90 countries signed the Stockholm Convention on Persistent Organic Pollutants, committing to eliminate the use of 12 POPs of greatest health concern. An exception was made for use of dichlorodiphenyltrichloroethane (DDT) for malaria control, and this use is increasing in many developing countries. Use of new compounds not covered under the treaty, such as polybrominated diphenyl ether (PBDE) flame retardants, is also increasing. Both DDT and PBDEs are endocrine disruptors and neurotoxicants in animals, but studies in humans are limited. Age at onset of puberty in girls, and possibly boys, has declined in recent decades. One hypothesis is that endocrine-disrupting compounds, including DDT or PBDEs, may be responsible for these trends. These pollutants may also jeopardize children's cognitive development, particularly with exposure during the prenatal and peri-adolescent periods of peak brain growth. We propose to investigate the associations of DDT and PBDE exposure in utero and at age 9 years on pubertal onset and neurodevelopment in the CHAMACOS longitudinal cohort of Mexican-American children in the Salinas Valley, California. The CHAMACOS children, born in 2000 and 2001, received relatively high in utero exposures to DDT from their mothers, who immigrated from areas in Mexico with recent DDT use. At 12 and 24 months of age, we found significant, dose-dependent deficits in cognitive functioning related to in utero DDT blood levels. Through their upbringing in California, CHAMACOS children likely have high exposure to PBDEs used in furniture and electronic products. As CHAMACOS girls approach age 9, we plan to enroll 130 new girls from the same source population as the original cohort, expanding the study from ~170 girls to 300. At the 9-year- old visit, we will collect blood samples from all 300 mother/daughter dyads and measure DDT and PBDEs. We will construct models to estimate in utero levels of DDT and PBDEs in those missing these measures, based on 9-year maternal levels and prenatal levels measured in 140 original participants. We will assess the relationship of in utero (measured or extrapolated) and age 9 (measured) blood concentrations of DDT/E and PBDE with the general cognitive, executive, attention, memory, and social cognition functioning of the girls assessed at ages 9, 101/2, or 12 years and with the timing and tempo of their pubertal onset as assessed by clinical Tanner exams conducted every six months from age 9 to 12 years. This application takes advantage of a rich dataset of Mexican-American children participating in the CHAMACOS longitudinal cohort study and proposes to expand this unique cohort. We aim to address key data gaps on the human health effects of these currently used compounds crucial for assessing their public health costs and benefits and necessary for national and international regulatory decision-making.

This project will develop statistical methods relevant to two common forms of environmental epidemiologic studies. The primary goal is to provide methods that extract a concise assessment of health risks associated with environmental exposures, supplemented by appropriate statistical inference. The first topic will evaluate the association between exposure and the risk of a health outcome using diagnosis data on a cohort of individuals supplemented with screening information on undiagnosed participants. The methods will be applied to data from the Seveso Women's Health Study which addresses health risks in women exposed to high levels of dioxin. It is intended that the statistical methods will apply generally to similar studies that include a combination of diagnostic and screening data. The second project concerns statistical techniques to investigate the effects of multiple environmental exposures on health and developmental outcomes. The ideas will be applied to data from the CHAMACOS study of Latino women and their children in California, where information has been collected on environmental (largely pesticide) exposures, in utero and in childhood, for a cohort of women and their infants. Statistical issues involve estimation and ranking-in importance-of suitable causal effects of each exposure, supplemented by a rigorous assessment of which of these represent real effects rather than spurious associations, allowing appropriately for multiple comparisons. Both studies involve the study of vulnerable populations exposed to above average environmental exposures with the potential for elevated risk for poor health outcomes. Statistical and computational algorithms will be developed and provided in an open source user-friendly format allowing their rapid dissemination and use by other investigators. The relevance to public health is two-fold: first, the research will allow environmental epidemiologists to accurately describe the effects of (i) acute dioxin exposure on the reproductive health of women, in particularly on the onset of fibroids, and of (ii) pesticide exposures on birth outcomes and subsequent neurodevelopment of children born to Latino women, in a farm working community. Second, the proposed research will provide appropriate statistical tools and software to allow other investigators to apply these complex methods to similar studies of the effects of environmental exposures in a wide variety of settings.