Betsy Lozoff - US grants

The proposed study will determine if iron therapy can correct rhe developmental deficits and behavioral abnormalities associated with iron deficiency anemia in infancey. The four aims of the project are: 1) to determine the level of iron depletion at which infant behavior is adversely affected, 2) to determine if there are differences between injectable and oral iron in behavioral effects, 3) to determine if iron therapy can completely correct the deficits, and 4) to confirm the specific pattern of behavioral disturbances observed in iron deficient anemic infants in our first study. The research design is a double-blind randomized controlled study of the short- and long-term behavioral and developmental effects of intramuscular and oral iron treatment. Two hundred one- to two-year old infants will be studies, 60 with iron deficiency anemia, 60 with iron deficiency with intermediate hemoglobin values, and 80 who are non-anemic, divided equally into iron depleted and iron replete groups. Their behavior and development will be assessed using sensitive quantitative behavior measures as well as overall tests of infant intelligence. To identify the level of iron depletion at which iron deficiency impairs infant development (Aim 1), pretreatment comparisons will be made in the groups of infants, whose iron status corresponds to the physiologic progression involved in becoming anemic from lack of iron. To accomplich (Aim 2,) the development and behavior of iron deficient infants treated with intramuscular iron will be compared to that of similar groups treated with oral iron, before treatment, after one week and after three months. Iron deficient infants will be compared with an iron replete control group before and after all babies become iron replete to determine if iron therapy can completely correct any behavioral deficits (Aim 3). Finally, (Aim 4) the study will confirm our previous results which demonstrated a specific pattern of impaired cognitive functions and a strong relationship between abnormal behavior and poor developmental test performance in iron deficient anemic infants. The results will guide practical pediatric and public health dexisions about preventive strategies, screening priorities, the level of iron deficiency requiring treatment, and the mode of therapy for iron deficiency anemia, the most common nutritional disorder in the world.

The proposed research will determine 1) if behavioral and developmental abnormalities associated with iron deficiency anemia during infancy can be corrected with extended iron therapy and 2) if deficits persist in school-aged children who had iron deficiency anemia as infants. In our current study, subjects whose iron deficiency and anemia were both corrected after three months of iron therapy also showed correction of their mental and motor test score deficits. Only 36% of all infants in the anemic group, however, showed this pattern of recovery; in the remaining 64%, biochemical evidence of iron lack and deficits in both mental and motor test scores were still evident even after the three-month course of oral iron or intramuscular iron therapy recommended for correction of the anemia for iron deficiency. To pursue these findings, two studies are proposed. Study I is a double-blind randomized controlled study of the behavioral and developmental effects of complete correction of iron deficiency and anemia in infancy. 90 one- to two-year-old infants will be studied: 30 iron deficient anemic infants, matched by age and sex with 30 nonanemic placebo-treated controls and 30 nonanemic iron-treated controls. Their behavior and development will be assessed before and after both three and six months of oral iron therapy. The study will also provide an opportunity to confirm the specific pattern of limitations, particularly in motor functioning, observed among iron deficient anemic infants in the current project. There are two major possible outcomes of the study: 1) an extended course of iron therapy will correct developmental and behavioral abnormalities in iron deficient anemic infants, a result that would require a major reformulation of current recommendations about iron therapy and monitoring of therapeutic response in infants or 2) pretreatment deficits will persist in some iron deficient anemic infants despite extended treatment, indicating that, depending on chronicity, severity, or timing, iron deficiency anemia in infancy may have irreversible effects. In Study II all 191 children in the current cohort will be reassessed at five years of age using psychoeducational, neurological, motor, and behavioral measures that focus on the specific functions that were identified as impaired in our previous two studies. The proposed research will determine the long-term cognitive, motor, and behavioral sequelae of iron deficiency anemia in infancy and the effects of treatment in a group of children which has already been exceptionally well characterized.

The long-range goal is to understand the behavioral and developmental effects of iron deficiency in infancy. Iron deficiency anemia affects one in 4-5 of the world's babies, and iron deficiency without anemia affects many more. The goal of the proposed 5-year project is to determine late functional consequences of early iron deficiency. This study continues to follow a well-maintained longitudinal cohort of 191 late adolescents/young adults in Costa Rica whose iron status and iron therapy were carefully documented in infancy. 87 percent of the original cohort is still participating. The project pursues our findings of major differences in school achievement and internalizing behavior problems at 10-13 years by assessing cognitive, motor, and emotional functioning at 19 years and the pursuit of higher education, job stability and level, mental health, early childbearing, etc. Late physical effects (delayed pubertal progression, obesity, stunting, cardiovascular risk factors) will be examined. Hypothesis-driven examination of specific cognitive functions will pursue earlier findings suggesting problems in spatial/working memory and selective attention/inhibition of the irrelevant. These functions are related to neural systems that have been shown to be altered in animal models of early iron deficiency. Sequential blood samples will again be obtained to extend earlier findings of stability in prolactin response from infancy to 12 years, relationships to behavior in early adolescence, and differing response depending on iron status in infancy. These assessments of specific cognitive functions and neuroendocrine responses to stress seek to identify the neural substrates underlying central nervous system effects of early iron deficiency. Beginning with longitudinal data already available from previous waves (infancy with 3 assessments, 5 years, 10-13 years, and 15-16 years), powerful statistical techniques such as hierarchical linear modeling and structural equation modeling will be used to identify patterns of continuity and change over time and to help clarify how biologic and environmental risks combine to result in poorer outcome.

The proposed project will 1) test the hypothesis that changes in neuromaturation observed in iron-deficient anemic infants are due to hypomyelination and 2) test a model of mechanisms explaining why iron-deficiency anemia in infancy is associated with poorer developmental outcome. Given the important role of iron in myelin formation and maintenance, impaired myelination is a promising explanation for recent evidence of immature neuromaturation in 6- month-olds with iron-deficiency anemia (slower Central Conduction Time, decreased vagal tone). The hypomyelination hypothesis (Aim 1) will be tested directly in a developmental rat model by examining vagal/optic nerve and regional brain myelination and indirectly in the child using auditory and visual evoked potentials and vagal tone measures from sleep studies. Annual neurophysiology evaluations will be conducted until children are 5 years of age to determine if the findings to date represent irregular progressions or arrests in neuromaturation (n=226, half initially anemic or nonanemic). The proposed model of the effects of early iron-deficiency anemia (Aim 2) postulates that hypomyelination and impaired dopaminergic function contribute to altered behavior in anemic infants, which interferes with their learning from the physical and social environment (functional isolation~) and makes them more vulnerable to the effects of environmental stressors, leading to poorer intellectual and motor development and more internalizing behaviors. Causal modeling techniques will be used to evaluate this model (n=1000 for testing the overall model; n=350 for exploring underlying mechanisms related to neuromaturation, dopaminergic function, and specific behavioral alterations). The proposed integration of a developmental animal model, related measures of CNS functioning and behavior in the child, and an overall model of explanatory mechanisms promises to be a major advance in understanding poorer developmental and behavioral outcome in early iron deficiency anemia, which affects 20-25% of the world~s babies and many poor or minority infants in the U.S.

Abstract

Frameworks for Integrating Nutritional, Environmental, and Neuroscience Contributions to Children's Development

T. D. Wachs & B. Lozoff


A workshop is planned to focus on the Functional Isolation Hypothesis. This hypothesis can serve as a theoretical framework for integrating the linked contributions of nutrition, environment, and neuroscience to children's cognitive and socio-emotional development. The core of the hypothesis is the assumption that nutritional deficits simultaneously act to adversely influence both central nervous system (CNS) development and function, as well as the level of the child's involvement with their environment and the nature of parent-child transactions. Parent-child transactions and the child's involvement with their environments, in turn, can impact the child's CNS development and functioning. Within this framework developmental outcomes are viewed as the result of the operation of linked nutritional, environmental, and CNS influences. Using the functional isolation hypothesis to link nutrition, environment, and neurosciences, a workshop is proposed to bring together 15 leading researchers from these 3 domains to discuss critical core questions that are central to the integrated study of functional isolation at the human level. The goal is to form research networks among nutritional, environmental, and neuroscience researchers that will focus on the interdisciplinary study of the nature of functional isolation and its consequences for children's development.

DESCRIPTION (provided by applicant): Approximately 20-25 percent of the world's babies have iron-deficiency anemia, and a much higher proportion has iron deficiency without anemia. The proposed 5-year project uses a brain/behavior framework to determine long-term effects of this common problem. The project's Specific Aims are to 1) establish whether neurophysiologic changes in iron-deficient anemic infants represent irregular progressions or arrests in neuromaturation; 2) identify long-term effects of preventing iron deficiency in infancy; 3) determine the effects of timing of iron-deficiency anemia in infancy on long-term outcome; and 4) characterize family/environmental factors that exacerbate or ameliorate poorer outcome. The project will reassess 1200 Chilean 10-year-olds who participated in NIH-supported studies as infants and preschoolers. Follow-up measures are based on new research on iron's role in the developing brain (effects on myelination, dopaminergic neurotransmission, and neuronal metabolism). A neuromaturation component uses advanced methods to assess sensory systems (ABRs, VEPs), spontaneous motor activity, sleep-wake cycle and autonomic nervous system, neurocognitive functions (with ERP), and neuroendocrine responses. The measures pursue findings of alterations in infancy and/or at 3-5 years. This component will involve 50 children who had iron-deficiency anemia at 6 months, 80 identified at 12 months. and 20 identified at 18 months; 50 children who were nonanemic at 6 months and received iron for a year will serve as the control group. The other component will determine the long-term effects of preventing iron deficiency in infancy. Evaluation of all 1200 children will pursue our findings of effects of iron supplementation on motor milestones, information processing. and behavior. The overall design supports analysis of outcome by age of iron-deficiency anemia to identify functions that are more or less affected, depending on developmental stage. and takes a comprehensive approach to environmental risk. The project promises to be a major advance in understanding this common nutrient deficiency and will inform policy decisions regarding the prevention and treatment of early iron deficiency in industrialized and developing countries alike.

Project I (human infant) focuses on brain-behavior effects depending on the timing of iron deficiency (ID) and iron repletion in human infants. Iron deficiency (ID) is the world's most common single nutrient disorder, differentially affecting pregnant women and infants everywhere. Project I promises to be the first systematic investigation of brain and behavior effects of prenatal dietary iron deficiency in human infants (Aim 1). The design will support comparisons of brain/behavior effects depending on the timing and duration of ID (Aim 2). The study will assess reversibility of effects, depending on timing of ID and its treatment (Aim 3), and examine maternal vs. fetal iron regulatory mechanisms in placenta and white blood cells (Aim 4). State-ofthe- art neurophysiologic and behavioral measures will test specific hypotheses regarding effects of ID on sensory, motor, cognitive, affective-social and regulatory functions related to impaired myelination of sensory/motor systems and altered structure, neurotransmitter function and neurometabolism in targeted brain regions (basal ganglia and hippocampus). The study will be conducted in China, a rapidly developing country where ID often occurs among pregnant women and infants in the absence of generalized undernutrition. Cord blood hemoglobin (Hb) andferritin concentrations will be measured in 1122 rural fullterm infants, with iron status determined again at 9 and 18 mo. Brain-behavior assessments in the perinatal period will involve 359 infants ("newborn cohort"): 59 with low Hb ("low birth iron" group) will receive iron;200 with marginal Hb or low cord ferritin ("marginal birth iron" group) will be randomly assigned at 6 wk, 50 to iron therapy and 150 to placebo;and 100 with normal cord Hb and ferritin levels ("normal birth iron" group) will receive placebo. The remaining 763 infants with cord blood testing will form the "blood screen cohort." At 9 and 18 mo, the newborn cohort will be reassessed, along with IDA infants from the blood screen cohort - about 58 at 9 mo ("early postnatal IDA") and 48 at 18 mo ("late postnatal IDA"). Approximately 39 marginalbirth- iron placebo-treated infants in the newborn cohort may also have IDA at 9 mo ("combined ID"). IDA infants will be treated with iron. Project I is tightly linked conceptually and methodologically with monkey and rodent projects in PPG2 and builds directly on findings in all projects in PPG1. Differential effects and/or reversibility depending on timing of ID or treatment could inform health policy and practice worldwide. However, the effects of prenatal iron deficiency have received very little study in human infants due in large part to previous thinking, no longer accepted, that the infant was protected. Up to 75% of pregnant women worldwide are anemic, with about half due to ID. An estimated 20-25% of 6- to 24-mo-old infants have IDA, and more have ID without anemia. Thus, the public health implications of study findings, especially in combination with Project II-IV animal models, could be profound.

developmental neurobiology; iron disorder; Primates; animal colony; clinical research;

[unreadable] DESCRIPTION (provided by applicant): The proposed 5-year project uses an integrated brain/behavior framework to determine long-term effects of iron deficiency anemia (IDA) in infancy. The project will evaluate approximately 1300 16-year-old Chilean adolescents, followed since infancy, including 200 who will receive sophisticated neurophysiologic assessments. Specific Aims are: 1) to test the hypothesis that early IDA will increase vulnerability to subsequent insults and stressors (i.e., "fragile recovery"), specifically adolescence with its dramatic brain maturation and expectable physical, social, and academic stressors; 2) to characterize interconnections among neuromaturation alterations and assess their functional significance; and 3) to identify genetic factors that increase susceptibility to iron deficiency and its effects. We expect that brain and behavior systems showing marked changes in adolescence, particularly those involving dopamine (DA) systems, will be particularly sensitive to early IDA (e.g., sleep-wake patterns, motor performance, neuroendocrine patterns, affective responses, reward system, and executive function) (Aim 1). We also predict that former IDA adolescents, who already show altered sleep-wake patterns, will have more social-emotional and neurocognitive disruptions with school day v. holiday/weekend cycle shifts. Neurophysiology studies will be performed for 200 subjects (half-day session for sensory evoked potentials & neurocognitive testing (some with ERP); 2 consecutive nights with polysomnography and sequential neuroendocrine sampling; 7-day activity monitoring at home). All 1300 subjects will receive a half-day test battery derived from our brain/behavior framework. For Aim 2, sleep-wake patterns, motor activity, heart rate and heart rate variability, and stress-response and night-time cortisol and prolactin patterns will be jointly analyzed. Development of the sophisticated data analytic methodology needed for this aim will start with neurophysiology data available for over 200 subjects at 10 years. The resulting methods will be applied to the 16-year data as they are completed. Aim 3 focuses on functional polymorphisms related to iron regulation (e.g., transferrin, HFE, etc.) and interactions between IDA and variants related to DA, 5-HT, anxiety/depression, ADHD, substance use and risk-taking (e.g., COMT, DRD4, 5-HTTLPR, etc.). All 1300 subjects are involved in order to detect gene-nutrition (IDA in infancy) interactions. The proposed study is the only long-term follow-up of a randomized trial of preventing IDA in infancy. The study will also continue to make unique contributions with its neuromaturation approach and will enter new territory by considering neurophysiologic regulatory processes and genetic vulnerabilities to iron deficiency and its effects. This study remains at the forefront of understanding long-term brain and behavioral effects of iron deficiency in the human infant. Iron deficiency is the world's most common single nutrient disorder; 20-25% of the world's infants have iron deficiency anemia and at least as many have iron deficiency without anemia. Long-term ill effects continuing into adolescence, despite iron therapy in infancy, could therefore have major public health implications. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Iron deficiency (ID) is the most common single nutrient disorder in the world, and pregnant women and infants are at highest risk. With long-lasting differences in prior studies, effects on the developing brain and infant behavior and development are among the most worrisome concerns. However, fundamental questions about causality, timing, duration, and severity of early ID remain unanswered. The proposed study will determine developmental/ behavioral effects of preventing ID depending on timing (Aim 1) and duration (Aim 2) of iron supplementation (i.e., pre- and/or early postnatally). The study will relate outcomes to severity of ID (Aim 3) and consider reversibility of effects with iron therapy, depending on timing (Aim 4). We expect different neurobehavioral effects when ID occurs or is prevented/treated during different phases of brain development (proliferation and growth phase primarily prenatally and regional diversification and interconnection largely in infancy). The project entails 2 randomized controlled trials (RCTs) to support causal inferences about preventing ID pre- and/or early postnatally. The project builds on a large CDC- supported study involving pregnant women in rural China. Study groups of infants in the proposed RCTs combined are a) pre- and early postnatal iron, b) prenatal iron, c) early postnatal iron, and d) neither (n = 500/group, total 2000, at study end). Iron status and sensitive sensory, motor, cognitive, language, and social-emotional outcomes will be assessed at birth, 6,12, and 24 mo. Comparisons of groups b) v. c) relative to d) will identify differential effects of preventing ID pre- v. early postnatally (Aim 1, timing). Comparisons of groups a) pre- &postnatal iron v. b) or c) will determine whether the duration of early iron supplementation has differential behavioral/developmental effects (Aim 2). Results of Aims 1 &2 will determine the best window to prevent ID effects and whether breast-fed infants benefit from iron before 6 mo. Infant iron status measures at each age will be analyzed individually and in combination to determine the level of ID at which different developmental domains are adversely affected (severity, Aim 3). If ill effects of ID without anemia are documented, there could be major policy implications;screening is currently only for anemia. Detecting more or less vulnerable domains may also point to other interventions in addition to iron therapy. To identify reversibile effects, depending on age of treatment (Aim 4), outcomes at 24 mo will be compared for infants 1) never iron-deficient, 2) poor iron status at birth and assigned to postnatal iron, 3) maternal IDA (anemia) treated in the 1st or 2nd trimester, 4) infant IDA treated at 6 mo, and 5) IDA treated at 12 mo. The study's comparisons of available approaches - iron supplementation and its timing in breast- fed infants, screening and treatment at different ages - will make the results highly relevant to global practice and policy regarding ID, which differentially affects poor and/or minority women and infants everywhere.

The overall purposes of this program-project grant (PPG) are to understand how iron deficiency alters brain and behavior in early development and identify interventions that will correct or prevent ill effects in the shortand long-term. Up to 75% of women worldwide are anemic during pregnancy, with about half due to iron deficiency. In developing countries, 46-66% of children <4 years are anemic, again with half attributed to iron deficiency, and iron deficiency disproportionately affects poor and/or minority mothers and infants everywhere. Yet there are still important unanswered questions about the brain and behavior effects of early iron deficiency. In the next 5 years, the PPG will focus on 1) timing of iron deficiency in relation to different stages of brain development, 2) timing of iron repletion to ameliorate short-term effects and prevent longterm consequences for brain and behavior, and 3) in-depth study of short- and long-term effects and the processes that account for them. PPG involves 4 projects (1 human infant, 2 monkey, 1 rodent) supported by 3 cores (administrative, analytical, and statistical).The component projects and cores, with interdisciplinary collaboration among leading clinical and basic science researchers, are tightly linked conceptually and methodologically, designed so that each has a special but complementary role. Project I (human infant) will be a systematic investigation of brain and behavior effects of pre- v. postnatal v. combined pre- and postnatal iron deficiency in human infants and the timing of iron treatment. Project II (Davis monkey) will use an experimental model to pursue its novel finding that prenatal iron deprivation produced a behavioral profile of reduced inhibition and increased impulsivity, despite iron repletion. Project III (Madison monkey) will assess brain-behavior effects of combined pre- and postnatal iron deficiency in infants born to young mothers - a naturalistic model directly relevant to vulnerable human populations (developing countries, adolescent mothers). Project IV (developing rodent) will focus on the effectiveness of iron therapy at different times in brain development in preventing genomic, biochemical, structural, and behavioral alterations in adulthood. It will also consider the potential for neurotoxicity with too much iron following early iron deficiency. With close integration, all projects assess neural and behavioral development. Each uses innovative approaches to assess the brain (e.g., electrophysiology in Project I, PET with drug challenge in Project II, brain tissue in Project III, and regional genomics in Project IV). Individually, each project represents a substantial leap beyond previous research on early iron deficiency. Collectively, the program will make major contributions to understanding, treating, and preventing brain and behavior effects of iron deficiency, the world's most common single nutrient disorder.

DESCRIPTION (provided by applicant): Recent studies using national data have reported that developmental disabilities are prevalent (as high as 1 in every 6 children) and on the rise in the US and elsewhere. While the cause(s) of these trends are unknown, there is concern for the potential role of exposures to environmental neurotoxicants. There is growing animal evidence that agents to which many or most people are exposed at varying levels, including contemporary-use pesticides, heavy metals, and secondhand tobacco smoke (STS), are neurotoxic. With the exception of lead, human studies of these agents in relation to neurodevelopment are lacking, both alone and together as mixtures. In many settings elevated exposure to these agents may be accompanied by nutritional deficiencies, with iron deficiency (ID) being the most common. This may result in greater neurotoxicity by acting through similar mechanistic pathways or increasing susceptibility to exposure. Pregnancy and infancy are the periods of greatest concern due to vulnerability of the developing brain. The proposed study aims to: 1) Investigate the association between neurodevelopment and early life exposure to pesticides, metals (manganese and lead), and STS individually and in combination (i.e., additive and multiplicative effects); 2) Test for interactions between these agents and ID on neurodevelopmental outcomes; and 3) Determine whether iron supplementation in pregnancy and/or in infancy ameliorates adverse impacts of environmental exposures on neurodevelopment outcome. The study will leverage two large ongoing NIH-supported studies of ID and neurodevelopment (n ~ 2500) taking place in rural China where elevated exposures, as well as widespread ID, have been documented. The study will utilize state-of-the-art approaches to assess exposures and outcomes. Detailed measures of sensory, motor, cognitive, affective-social and regulatory functions at birth/6 weeks, 9 months, and 18 months of age are collected in the ongoing studies. Exposure biomarkers will be measured in urine and blood samples from multiple time points in early development, including sensitive new methods to quantify >45 pesticides in cord blood. Expected outcomes of this study are to provide new and much needed information on the impacts of environmental exposures on neurodevelopment and prioritization of exposure risks, the specific processes and sensitive exposure windows involved the simultaneous effects of multiple exposures and ID on these endpoints, and evidence for potential interventions to lessen these effects through exposure reduction or iron supplementation. The study will likely have a significant impact on public health policy given widespread exposure to these agents worldwide, the global prevalence of ID, and increasing evidence that many environmental exposures contribute to poorer neurodevelopmental outcomes.

DESCRIPTION (provided by applicant): Recent studies using national data have reported that developmental disabilities are prevalent (as high as 1 in every 6 children) and on the rise in the US and elsewhere. While the cause(s) of these trends are unknown, there is concern for the potential role of exposures to environmental neurotoxicants. There is growing animal evidence that agents to which many or most people are exposed at varying levels, including contemporary-use pesticides, heavy metals, and secondhand tobacco smoke (STS), are neurotoxic. With the exception of lead, human studies of these agents in relation to neurodevelopment are lacking, both alone and together as mixtures. In many settings elevated exposure to these agents may be accompanied by nutritional deficiencies, with iron deficiency (ID) being the most common. This may result in greater neurotoxicity by acting through similar mechanistic pathways or increasing susceptibility to exposure. Pregnancy and infancy are the periods of greatest concern due to vulnerability of the developing brain. The proposed study aims to: 1) Investigate the association between neurodevelopment and early life exposure to pesticides, metals (manganese and lead), and STS individually and in combination (i.e., additive and multiplicative effects); 2) Test for interactions between these agents and ID on neurodevelopmental outcomes; and 3) Determine whether iron supplementation in pregnancy and/or in infancy ameliorates adverse impacts of environmental exposures on neurodevelopment outcome. The study will leverage two large ongoing NIH-supported studies of ID and neurodevelopment (n ~ 2500) taking place in rural China where elevated exposures, as well as widespread ID, have been documented. The study will utilize state-of-the-art approaches to assess exposures and outcomes. Detailed measures of sensory, motor, cognitive, affective-social and regulatory functions at birth/6 weeks, 9 months, and 18 months of age are collected in the ongoing studies. Exposure biomarkers will be measured in urine and blood samples from multiple time points in early development, including sensitive new methods to quantify >45 pesticides in cord blood. Expected outcomes of this study are to provide new and much needed information on the impacts of environmental exposures on neurodevelopment and prioritization of exposure risks, the specific processes and sensitive exposure windows involved the simultaneous effects of multiple exposures and ID on these endpoints, and evidence for potential interventions to lessen these effects through exposure reduction or iron supplementation. The study will likely have a significant impact on public health policy given widespread exposure to these agents worldwide, the global prevalence of ID, and increasing evidence that many environmental exposures contribute to poorer neurodevelopmental outcomes.

DESCRIPTION (provided by applicant): Epigenetic effects are reported for prenatal environmental exposures such as lead (Pb), pesticides, bisphenol A, and stress and nutrient deficiencies, such as folate or iron deficiency (ID). Most studies consider single exposures. However, exposure to mixtures of environmental toxicants, nutrient deficiencies or harmful dietary additives, and stressors is common. This pilot study will explore epigenetic changes and neurodevelopmental outcome with real-life prenatal exposures (mixtures of pesticides, Pb, and ID). The overarching hypothesis is that exposure mixtures are associated with changes in offspring DNA methylation patterns of key neurodevelopmental genes/pathways related to poorer neurocognitive function later in life. The pilot study builds on NIH-funded projects on neurodevelopmental impacts of pre- and postnatal environmental exposures and ID, involving cohorts from rural areas near Beijing and Hangzhou, China (2000 full-term infants). The infant studies share the same brain-behavior conceptual framework. Parent study measures of environmental exposures, iron status, growth, behavior, sensory systems, and motor and cognitive function were collected at birth/6 weeks, 9 and 18 months. Over 200 pesticides are assayed in cord blood. Cord blood samples have been saved frozen for genetic analyses. To identify epigenetically modified genes related to exposures and neurodevelopment, we propose a genome-wide DNA methylation approach, with the following Specific Aims: 1) To demonstrate feasibility of epigenetic studies using Illumina Infinium HumanMethylation450 BeadChip analysis in these cohorts in China; 2) To identify genome-wide DNA methylation changes associated with prenatal exposure mixtures; and 3) To identify epigenetic changes that may mediate the association of exposures with markers of neurodevelopment. In selecting 96 cord-blood samples from each cohort for DNA- methylation studies (total n = 192), we prioritized unique features of the parent study designs: highly sophisticated, innovative brain-based measures for Hangzhou and environmental exposure measurement during pregnancy for Beijing. We chose specific outcomes where neural processes are well-characterized in animal models: ABR (myelination), recognition memory with ERP (hippocampus), and sequence learning (basal ganglia). Per China regulations, epigenetic assays will be conducted in China. Bioinformatic and biostatistical data analysis and interpretation will occur at the University of Michigan. We plan future studies using the entire pool of 2000 infants to disentangle epigenetic modifications with pesticides, Pb, and ID; consider exposure timing; validate DNA methylation profiles quantitatively; and determine epigenetic-exposure- outcome relations that can be modified by nutritional intervention, with iron supplementation as an illustration.