Methylmercury (MeHg) exposure from fish consumption during childhood may adversely affect developmental outcomes. The brain is not fully developed until halfway through the third decade of life and is therefore potentially susceptible to MeHg exposure well beyond the fetal period. It is currently not known if it is safe for children to eat fish. While epidemiological evidence supports the hypothesis that postnatal MeHg exposure is adversely associated with children's development, studies have not been specifically designed to study postnatal MeHg exposure. Nearly all studies have been cross-sectional evaluations in which MeHg exposure was measured only at the time of cognitive testing (convenience samples). No studies have employed the more definitive and powerful approach of longitudinal assessment of postnatal MeHg exposure in relation to cognitive outcomes throughout childhood and adolescence. We are in a position to study this issue longitudinally in the Republic of Seychelles where dietary MeHg exposure is among the highest in the world. In 1989-90 we enrolled a prospective cohort of 779 mother-infant pairs (“?Main Cohort”?). We have 24 years of postnatal exposure and neurodevelopmental data in this cohort, which has been characterized more extensively than any other cohort. In cross-sectional analyses we have found adverse associations between recent postnatal MeHg exposure and various developmental endpoints at different ages, but especially with measures of psychomotor and executive function, attention, and general intelligence at 9 years of age and older. These cognitive domains have their greatest development postnatally and are therefore more likely to be affected by postnatal MeHg exposure. These intriguing results need to be studied using longitudinal analyses that better address the variability in postnatal exposure and its impact on neurodevelopment over time. The aim of this proposed study is to examine the longitudinal associations of cumulative postnatal MeHg exposure with neurodevelopment. We hypothesize that greater accumulated postnatal exposure is adversely associated with neurodevelopmental outcomes in later childhood and adolescence. We will use a previously-developed cumulative exposure metric based on repeated measurement of children's hair MeHg values. So far, this metric has only been examined in relation to one developmental outcome at 9 years of age. We now have an additional 15 years of exposure and developmental data on this cohort. We will leverage these data and examine three cumulative postnatal exposure metrics (early life, late adolescent, and lifetime) in relation to cognitive domains that have shown adverse associations in cross-sectional analyses. Longitudinal models will evaluate the consistency of associations over time. Billions of people depend daily on the nutritional properties of fish. It is therefore critical to examine whether children's exposure to MeHg from consumption of fish is associated with adverse developmental consequences. This study will provide the most comprehensive assessment to date of the risks or safety of fish consumption in childhood.

Fish is a major protein source and a primary source of nutrients such as long chain polyunsaturated fatty acids (PUFA) which are essential for maternal and fetal health. All fish also contain methylmercury (MeHg), a known neurotoxicant at high levels of exposure as demonstrated by accidental poisonings in Japan and Iraq. However, despite many years of research, there is still substantial uncertainty regarding the safety of eating fish with natural background levels of MeHg during pregnancy. Identifying the potential reasons for this uncertainty is important, as limited understanding of factors influencing MeHg toxicity represents a challenge for promulgating public policies on fish consumption. Our studies in the Republic of Seychelles have revealed that the association between prenatal MeHg exposure from maternal fish consumption and child developmental outcomes is far more complex than previously anticipated, and likely involve concomitant dietary exposures and genetic factors influencing MeHg toxicity. To examine these complex relationships, we enrolled a cohort of 1,536 mother-child pairs and characterized them for prenatal MeHg exposure, maternal nutritional status, and developmental outcomes at 20 months of age. Further, we examined the modifying role of genetic factors on MeHg metabolism and toxicity in our human cohort and in a Drosophila model. While we continue to find that MeHg does not influence neurodevelopmental outcomes independent of nutritional status, our epidemiological and experimental genetics findings support a novel biological framework that describes the role of GSH-dependent pathways and inflammation in MeHg toxicity. We plan to examine this framework, and will continue both developmental follow-up of the children and the exploration of nutritional and genetic components. We aim to evaluate how prenatal nutritional factors affecting inflammation, and genetic factors affecting GSH-dependent pathways influence associations between prenatal MeHg exposure and developmental outcomes. We hypothesize that the association between prenatal MeHg exposure from fish consumption and developmental outcomes is largely influenced by endogenous "protective metabolic pathways" (toxicokinetics) and exogenous dietary factors (toxicokinetics, toxicodynamics). We will re-examine the children at seven years of age for developmental outcomes, and will draw upon stored blood samples from both mothers and child's cord to investigate additional genetic factors involved in MeHg toxicokinetics and toxicodynamics, and measure inflammatory biomarkers as indicators of the role of the maternal inflammatory milieu in modifying MeHg toxicity. We plan to use experimental genetic studies to confirm the associations observed in our cohort and generate new hypotheses that can then be studied with an epidemiologic approach. DNA genotyping was determined for mothers and offspring. Polymorphisms of genes important to transfer of MeHg across the blood-brain barrier (ATP binding cassette family C: ABC) and for the metabolism of PUFA (fatty acid desaturase: FADS) were associated with maternal MeHg and maternal PUFA levels but not clearly with developmental endpoints. The project also includes a series of experiments using the Drosophila model which will examine the role of ABC and glutathione S-Transporters: GST genes for MeHg tolerance. Our approach will address novel hypotheses that should bring clarity to the interpretation of previous cohort studies, but also assist public policy makers in crafting advice to women of child-bearing age regarding the safety of consuming fish during pregnancy.

Methylmercury (MeHg) is a persistent environmental toxin that preferentially targets the developing nervous system. MeHg exposure is of great concern for human health due to its presence in dietary fish. However, the risks of MeHg exposure commonly encountered today remain uncertain, largely due to the variable outcomes observed among individuals experiencing similar levels of MeHg exposure. Our long-term objective is to resolve the molecular and genetic mechanisms that control MeHg toxicity and thereby contribute to more scientifically sound risk assessment. Our overall aim is to investigate a genetic component for susceptibility and tolerance to MeHg. In a screen for genes that confer tolerance to MeHg during development in Drosophila, we have identified cytochrome p450 (CYP) family members as candidates. CYPs are Phase I metabolism enzymes that modify xenobiotics in detoxification pathways and perform essential activating reactions for endogenous and exogenous biomolecules. We found CYP6g1 is the most highly expressed and regulated CYP in MeHg-tolerant flies and that over-expression of CYP6g1 by transgene or via a naturally occurring high-expressing allele confers MeHg tolerance. We therefore propose a novel hypothesis that predisposition to MeHg toxicity is influenced by innate levels of CYP activity. With this proposal we intend to improve scientific knowledge by translating our findings in flies to humans. Our two Specific Aims are designed to investigate the functional and genetic association of the human homologs of CYP6g1 (CYP3A4, CYP3A5 and CYP3A7) with tolerance to MeHg toxicity in people. CYP3A members are the most highly expressed CYPs in the human liver. CYP3A7 and CYP3A4 are distinguished by being the predominant fetal and adult CYPs, respectively. In addition, CYP3A4, CYP 3A5 and CYP 3A7 polymorphisms and haplotypes exist that are predicted to be more and less active, and which show high frequency Africans versus Caucasians, respectively. In Aim1 we will determine the relative activity of each of the CYP3A isozymes in conferring MeHg tolerance during development. This will be accomplished using our established MeHg-tolerance assay in transgenic Drosophila. In Aim2 we will probe for the association of CYP3A genotypes and MeHg toxicity outcomes in two established cohorts of MeHg-exposed children that are predominantly African or Caucasian. Our CYP single nucleotide polymorphism (SNP) probe analyses will be done as an addendum to an ongoing large-scale genotyping project with these two cohorts. Association of MeHg outcomes with CYP genotype will be resolved through multivariate analyses of CYP alleles with consideration of potential confounders. Results from this study will have a potentially lasting impact by identifying a genetic component of variability in susceptibility to MeHg in developing children. Identification of CYPs as genetic markers of susceptibility to MeHg would greatly advance risk assessment by bringing new technical abilities to health care providers for identifying and advising at-risk individuals in a clinical setting.

This application is a competing renewal of Grant 5-R01ES008442 entitled "Methyl Mercury Effects on Adolescent Development." The Seychelles Child development Study (SCDS) has been testing the hypothesis that methyl mercury (MeHg) exposure from consumption of a diet high in fish is associated with adverse health outcomes. Exposure to MeHg from fish consumption is thought to impair cognition and neuro-regulation of the heart. Since 1989, we have been following a cohort of 779 subjects born to mothers who consumed an average of 12 fish-meals per week during pregnancy. The subjects are now approaching 21 years of age. The subjects have been assessed for cognitive and behavioral development. In 2006, the project was expanded with funds from a competing supplement to include measurements of autonomic heart regulation. The subjects themselves habitually consume a diet high in fish. Fish contain both MeHg and nutrients including long-chain polyunsaturated fatty acids (LCPUFA) which are beneficial to brain development and heart function. Preliminary data suggest that prenatal and postnatal exposures to combined MeHg and LCPUFA in fish may be associated with distinct patterns of results. Limited information is available on postnatal exposures however. We will examine the SCDS cohort at approximately 21 and 23 years of age to assess the net cumulative risk of exposure to postnatal MeHg and recent LCPUFA status on cognitive outcomes and cardiovascular parameters. Statistical analyses will employ innovative parametric and non-parametric modeling designed to ascertain the effects on these endpoints of co-exposures to postnatal, adjusted for prenatal MeHg exposure, and/or lifetime cumulative (prenatal and postnatal) exposure to MeHg, and recent exposure to LCPUFA to ascertain the separate risks attendant to these exposures. The Seychelles population has many similarities with that of the US and can therefore serve as a sentinel for the risks and benefits of fish consumption. The low loss to follow-up provides a unique opportunity to continue to study this very well characterized cohort and differentiate prenatal from postnatal MeHg effects as its members move from adolescence to young adult life. The findings from this study will be important as governmental agencies continue to evaluate the scientific data regarding toxic effects and nutrient benefits of a high fish diet. The study should also clarify the risks and benefits of fish consumption in relation to neurocognitive and cardiac morbidity that may not become clinically manifest until later in adulthood.

Mercury (Hg) vapor and monomethylmercury (MeHg) are separately potent neurotoxins. Dental amalgam restorations, which are composed of approximately 50% metallic Hg, are the principal source of human exposure to Hg vapor, while most exposure to MeHg comes from fish consumption. Both forms of Hg cross the placental and blood-brain barriers, exposing the developing fetus. Of significant concern, the human fetus appears especially vulnerable to MeHg. There is a paucity of data on the developmental effects of prenatal Hg vapor exposure, and no data on the combined exposure to Hg vapor and MeHg exposure, despite concerns that combined exposure may elevate risk via additive or synergistic mechanisms. The proposed study will build on the strength of the Seychelles Child Development Nutrition Study (SCDNS), a longitudinal cohort study examining the association between prenatal exposure to MeHg, nutritional status, and development. Residents of the Seychelles have free dental care using amalgams and consume large quantities of ocean fish. SCDNS children have been evaluated eight times and their nutritional and developmental status to date has been well characterized. Prenatal and postnatal exposure to MeHg to date has been determined by periodic sampling of the mother's/child's hair and maternal dental status during pregnancy was established by oral examinations following delivery. In the proposed study, we will conduct routine dental exams in children recording amalgam restorations placed/present/lost as a measure of postnatal Hg vapor exposure and hair analysis as a measure of postnatal MeHg exposure. Neurodevelopmental testing will occur at 72 and 96 months. Preliminary data from a prior Seychelles cohort suggests an adverse association when Hg vapor exposure (as measured by maternal amalgams present during gestation) is included in analyses. The aim of the proposed project is to capitalize on the availability of the already well-characterized SCDNS cohort to quantify the level of risk for adverse neurodevelopmental outcomes attributable to prenatal and postnatal co-exposure to Hg vapor and MeHg. Three hypotheses will be tested: 1) The level of risk for adverse neurodevelopmental outcomes will be accentuated by combined exposure to Hg vapor and MeHg; 2) Effects of prenatal exposure will be accentuated by postnatal exposure; and 3) Overall exposure effects will be modulated by prenatal nutritional factors.