Although increasing animal evidence supports the hypothesis that in utero exposure to endocrine disrupting compounds can have a long-term impact on the health of the 2nd and subsequent generations, the evidence in humans is nearly non-existent. In addition, individuals may have differences in susceptibility to chemical exposure based on their genetic make-up. We propose to study genetic susceptibility in a unique population of children whose mothers, participants of the Seveso Women's Health Study (SWHS), were exposed to one of the most potent endocrine disruptors, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD or dioxin). On July 10, 1976, an explosion at a chemical plant near Seveso, Italy resulted in a toxic plume that exposed nearby residents to high levels of TCDD. The SWHS, a retrospective cohort study, was initiated in 1996, to investigate the health of 981 women who were newborn to age 40 years in 1976, had resided in the immediate vicinity of the plant, and had archived samples of blood collected soon after the explosion. The SWHS is the only comprehensive study of health effects of TCDD exposure in a female population, and has the unique benefit of measurements of individual-level TCDD in blood collected near the time of the explosion. We are currently re-contacting SWHS women to enroll ~955 children aged 0-38 years in a study of their health, to determine whether in utero TCDD exposure is associated with health outcomes at birth or later in life. The proposed study will use DNA from blood collected during the current (2014-2016) follow up to genotype mother-child pairs in the aryl hydrocarbon receptor (gene: AhR, protein: AhR) pathway, an enzymatic network that directs the metabolism of TCDD and other xenobiotics in humans. We will conduct a gene-by- environment (GxE) analysis to evaluate the modifying effect of genetic polymorphisms in the AhR pathway on susceptibility to dioxin toxicity in women and their children born after the explosion. In particular, we will use longitudinal measurements of serum TCDD (1976 and 1996) to examine differences in TCDD metabolism across genetic subgroups of the SWHS (Aim 1). We will then use genotype data on mother-child pairs in AhR pathway genes to examine interaction between the maternal and child genotypes with in utero dioxin exposure on birthweight in children born after the explosion. With its targeted focus on the AhR pathway, this study is uniquely equipped to examine the influence of genotype on dioxin half-life longitudinally and the first study of how both maternal and child genetics may shape fetal susceptibilities to dioxin.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), a widespread environmental contaminant, is a potent endocrine disruptor in animal studies. On July 10, 1976, as a result of a chemical plant explosion, residents of Seveso, Italy experienced the highest levels of TCDD exposure in a human population. In 1996, we initiated the Seveso Women's Health Study (SWHS), a retrospective cohort study of the health of 981 women who were newborn to 40 years at the time of the explosion, lived in the most contaminated areas, and had archived blood collected soon after the explosion. Individual TCDD level was measured in the 1976 archived serum, and for a subset, TCDD and total dioxin toxic equivalents (TEQ) were also measured in serum collected in 1996. In 2008, we followed-up the SWHS cohort to study potential longer-term health sequelae. The SWHS is the only comprehensive study to date of the health of a female population exposed to TCDD; it is unique in being a large cohort with a wide range of TCDD exposure, documented by individual serum TCDD measurements. Over the last 15 years, we have reported numerous findings on this 1st generation of TCDD-exposed women. We found that TCDD serum levels were associated with infertility and higher rates of cancer. However, some of the most profound associations were found among the women who were youngest at the time of the explosion. In this group, we found that TCDD serum levels were related to early menarche, increased menstrual cycle length, and increased risk of metabolic syndrome. Animal studies suggest those exposed in utero may be even more susceptible to the effects of TCDD, and that effects may persist through multiple generations. However, few human studies have examined 2nd generation health effects of TCDD. Now, nearly 40 years after the explosion, the SWHS cohort will have completed their families. We propose to re-contact the SWHS women and enumerate their children born after the explosion. We will invite all children ¿2 years to participate in a study of their health, and we will measure anthropometrics, blood pressure and clinical chemistries. We will also interview the mother and/or the child. For this initial 2nd generation study, we focus on the endpoints that were the most sensitive to exposure in the 1st generation; namely, we will determine whether in utero TCDD exposure is associated with obesity and metabolic syndrome, thyroid function, age at menarche, menstrual cycle characteristics, and infertility. We hypothesize that the 2nd generation will be as, if not more, susceptible than the 1st to the effects of TCDD exposure. This initial study will lay the foundation for the ongoing investigation of the long-term health of this 2nd generation and will begin to enumerate the 3rd generation. Although increasing animal evidence supports the hypothesis that in utero exposure to endocrine disrupting compounds can have a long-term impact on the health of the 2nd and subsequent generations, the evidence in humans is nearly non-existent. This study will be one of the first to examine the hypothesis of fetal origins of adult disease in a human population.

Over the last few decades, concern has heightened over the potential hormone-like effects of environmental chemicals on the reproductive health of fish, wildlife, and humans. The compound 2,3,7,8- tetrachlorodibenzo-p-dioxin (TCDD) is a widespread environmental contaminant, produced by various chemical reactions and combustion processes, and in animals is a potent endocrine disrupter. On July 10, 1976, a chemical explosion near Seveso, Italy exposed the residents to the highest levels of TCDD exposure in a human population. We followed up the women 20 years after the accident to determine whether they were at higher risk for reproductive disease. Women aged 0 to 40 years at the time of the explosion, and who lived in Zones A or B were interviewed and examined, and serum specimens stored from the time of the explosion were analyzed to determine individual levels of TCDD exposure. The Seveso Women's Health Study, originally funded in 1996 (R01 ES07171) aimed to examine the relationship of serum TCDD levels and endometriosis. Continued funding in 1999 and 2001 allowed the analysis of additional serum specimens for TCDD and examination of the relation to other reproductive endpoints: menstrual cycle characteristics, age of menarche, fetal loss and birth weight, age at menopause, breast cancer incidence, length of lactation, ovarian disease and fibroids. We found that the youngest women (< 10 years in 1976) were the highest exposed, but they were also the least likely to have experienced reproduction at follow-up (25 percent nulliparous in 1996). Among other findings, we observed a significant increased risk for breast cancer, but the cohort averaged only 40 years old in 1996. In this proposal, we aim to follow-up this unique cohort approximately 30 years after the explosion, when the women have aged and longer-term sequelae of exposure to TCDD may emerge. We expand our health assessment to include non-reproductive health outcomes likely affected by endocrine disruption. Specifically, we aim to study the relation of serum TCDD to breast cancer, diabetes and metabolic syndrome, bone density, and women and neonatal thyroid hormone. The proposed study takes advantage of previously collected data and specimens, including the 1976 serum TCDD levels and 1996 archived bloods. We propose to interview the women about their health and obtain medical records, and on a subsample, perform a bone density evaluation, and measure thyroid hormone and TCDD/TEQ in archived 1996 blood. We will acquire from registry records the neonatal TSH levels of all children born after 1994. The Seveso Women's Health Study is the only comprehensive study to date of the health of women exposed to TCDD. This study has the benefit of being a large cohort study with a wide range of TCDD exposure, documented by individual-level TCDD measured in sera collected soon after the explosion.

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.