Final Report: Neurodevelopment and Improving Children's Health following EtS exposure (NICHES)

EPA Grant Number: R835437
Center: The Center for Study of Neurodevelopment and Improving Children's Health
Center Director: Murphy, Susan K.
Title: Neurodevelopment and Improving Children's Health following EtS exposure (NICHES)
Investigators: Murphy, Susan K.
Institution: Duke University
EPA Project Officer: Nolt-Helms, Cynthia
Project Period: June 1, 2013 through May 31, 2018 (Extended to May 31, 2019)
Project Amount: $3,907,780
RFA: Children's Environmental Health and Disease Prevention Research Centers (with NIEHS) (2012) RFA Text |  Recipients Lists
Research Category: Children's Health , Health

Objective:

Project 1 will examine the relationship between smoke exposure during early life and neurobehavioral outcomes in children followed from prior to birth through up to age 7 years, with a particular focus on attention deficit / hyperactivity disorder (ADHD). This project will also examine the relationship between smoke exposure, ADHD and DNA methylation.

Project 2 will determine how exposure during early development to tobacco smoke extract and to nicotine influences growth and neurobehavioral outcomes in rats and neural differentiation and neurotransmitter phenotypes in vitro. This project will also work to define the most sensitive developmental window(s) of vulnerability to tobacco smoke and nicotine exposure and will determine if dietary interventions can ameliorate the effects of these exposures.

Project 3 will investigate how in utero tobacco smoke and nicotine exposure in rats influences DNA methylation in the brain and blood, and how the methylation profiles in the brain relate to gene expression. These findings will be applied to study of cord blood from our human cohort to determine if methylation patterns can be used to stratify risk of ADHD prior to onset of symptoms. ADHD-associated genes will also be examined in the in vitro models of neurodifferentiation and neurotransmission to determine associations with these phenotypes.

Our Community Outreach and Translation Core will develop an educational primer about the effects of tobacco smoke on children with particular emphasis on ADHD. The primer will be used as a tool to elicit interest in community participation in an Instagram contest that requires that the entry reflect what has been learned from the educational primer.

Conclusions:

OVERALL:Center for Study of Neurodevelopment and Improving Children's Health following EtS exposure (NICHES)

The NICHES Children's Environmental Health and Disease Prevention Research Center accomplished the overwhelming majority of its overall objectives, from a scientific standpoint, community outreach, and training. Scientific accomplishments are detailed under the respective sections of the report. We have had two faculty development investigators on the project. The first, Dr. Brandon Hall, a postdoctoral researcher working on Project 2, has taken a permanent position in Research Development in the Department of Surgery at Duke University School of Medicine. Dr. Julia Schechter replaced Dr. Hall in 2016 as our FDI. She was then a postdoctoral researcher working on Project 1 with Dr. Kollins and subsequently was offered and accepted a position as assistant professor in the Department of Psychiatry and Behavioral Sciences at Duke University Medical Center. Ms. Rashmi Joglekar has completed her doctoral thesis research working on NICHES, and we have had numerous other trainees, including rotating graduate students, Duke undergraduates, hosted and trained a visiting graduate student from the CHAMACOS cohort at Berkeley, and high school students who have devoted time to NICHES while receiving training in the methodologies and science that forms the foundation for the center's activities.

We created and maintain a web site (http://niches.duke.edu) that describes the NICHES projects, COTC and investigators. The web site has pages describing ADHD, Tobacco exposure and Epigenetics along with pages devoted to publications coming from the project, presentations, news, an information page and a page for contact information. A white board animation was created and placed prominently on the home page of the NICHES web site to convey the overall problem with tobacco smoke exposure during pregnancy and how the NICHES center was going to approach the questions that arise as a result. The NICHES Twitter feed is also displayed on the home page of the web site, as are links to the EPA, NIEHS, and other relevant web sites.

NICHES has published 52 articles, editorials or book chapters to date with more in process. We have established an informational web site, educational brochure and materials for public education that conveys the message that exposure to tobacco smoke should be avoided during pregnancy since it can harm the baby's genes. NICHES investigators have presented scientific findings or educational information related to NICHES at 74 meetings, conferences, webinars or symposia over the duration of funding.

NICHES established a Quality Assurance Project Plan at the project outset that was monitored annually by our Quality Assurance Manager, Ms. Sunita Patil. Ms. Patil was contracted through the Duke Office of Clinical Research and performed regular audits each of the three projects. As one component of our QAPP, we established a secured online server space in which all final data from the NICHES project is stored. Access to this server is restricted to those personnel working on the project.

Overall, NICHES has made strides in improving understanding of the potential biologic, neurologic, and behavioral consequences of tobacco smoke exposure during pregnancy and has revealed new information about one potential mechanism that mediates the link between exposure and neurobehavioral outcomes. Of particular relevance, our epigenetic data have identified a major enrichment of both autism and ADHD candidate genes that exhibit altered methylation in association with exposure to tobacco smoke in utero, both in rats and in humans. These findings are presented more fully under Project 3 but will provide strong impetus for future research to better understand these links.

PROJECT 1: Tobacco Smoke Exposure, Epigenetics and Cognitive Deficits in Children

Investigator(s): Scott H. Kollins, PhD, Bernard F. Fuemmeler, PhD, Cathrine Hoyo, PhD

Overview:

Prenatal and postnatal environmental tobacco smoke (ETS) exposure have been associated with a range of adverse cognitive and neurobehavioral outcomes in children, including higher rates of Attention-Deficit/Hyperactivity Disorder (ADHD). However, our understanding of both the developmental timing of ETS induced effects on cognitive outcomes, as well as the possible mechanisms underlying such effects is limited. Project 1 aimed to fill this gap by evaluating the associations of both ETS exposure on cognitive and neurobehavioral outcomes across early development and examining the role of exposure-induced DNA methylation changes on these outcomes. Specifically, the aims of Project 1 were: (1) To characterize the extent and developmental timing of ETS exposure effects on cognitive and neurobehavioral outcomes in young children; and (2) To determine the relation between DNA methylation and cognitive and neurobehavioral outcomes in young children. Our central hypotheses were that (1) pre- and postnatal ETS exposure will be associated cognitive functioning (e.g., executive function, attention) and neurobehavioral phenotypes (e.g., ADHD symptoms) and (2) that DNA methylation will be relevant to explaining the association between ETS exposure and cognitive and neurobehavioral outcomes. Ultimately, the study is the first of its kind to help disentangle the associations between ETS and childhood cognitive outcomes by exploring potential epigenetic factors that may help explain these associations. Because DNA methylation is malleable, the findings may inform novel methods for improving cognitive deficits resulting from ETS.

The study leveraged data from a perinatal birth cohort that obtained prospectively collected data from the first trimester through infancy including surveys regarding smoking history, data from medical records, maternal blood, and cord blood and buccal cells at delivery. As part of Project 1, detailed assessments were obtained of childhood cognitive, neurobehavioral function, and ADHD symptoms among a subcohort of children (N=350) at two time points, two years apart. Prenatal smoke exposure was measured via plasma cotinine collected from maternal blood specimens during pregnancy and ETS exposure was measured via saliva and blood collected from children during the Project 1 visits. DNA methylation for select regulatory control regions for genes that have been associated with ADHD symptoms or similar neurodevelopmental phenotypes were characterized from the child's cord blood and their peripheral blood collected during Project 1 visits.

Summary of Data Collected:

As of May 31st 2019, 350 participants had enrolled (Time 1) in NICHES Project 1. Two hundred and two of these participants returned to the lab and participated in the 2-year follow-up visit (Time 2). The sample was diverse (approximately 60% African American, 32% Caucasian, 5.8% multi-racial, 1.3% Asian, and 0.3% other or unknown race) and reflected a broad developmental period, with the mean age for children at the time of our enrollment of 5.6 years (SD=2.4, range=2-13) and the mean age for children at Time 2 of 7.1 years (SD=1.8, range=4-13). We completed baseline neurodevelopmental assessments at Time 1 and again at Time 2. For both mothers and their children, this included assessments of executive functioning using the NIH toolbox and an IQ test. We collected blood and saliva from these participants and a batch of the saliva specimens have already been analyzed for cotinine to objectively measure secondhand smoke exposure in the children. We plan to run additional analyses on the saliva samples as well as on the blood samples to examine potential DNA methylation in selected differentially methylated regions of interest. Loci chosen will be informed through putative pathways, recent findings in the literature, and findings from Project 2 and 3. The main goal of these analyses are to assess stability of epigenetic loci related to smoke exposure or predictive of attention and cognition from birth through childhood.

Our battery of neurodevelopmental tests for the child and mother participants included the NIH Toolbox. In addition, the Differential Abilities Scale, Second Edition (DAS-II) was administered to the child and the Wechsler Abbreviated Scale of Intelligence –II (WASI) was administered to the mother. Mothers also completed self-report measures to report on their own behaviors and those of their child. Measures included the Conner's Adult ADHD Rating Scales (CAARS), Strengths and Weaknesses of ADHD Symptoms and Normal Behavior Rating Scale (SWAN), Strengths and Difficulties Questionnaire (SDQ), Behavior rating Inventory of Executive Function (BRIEF), Behavior Assessment System for Children (BASC) and Parent Stress Index (PSI). The BASC and BRIEF were also completed by children's teachers. Taken together, these data will allow us to examine the longitudinal effects of smoke exposure on neurodevelopmental outcomes as well as behavioral and emotional functioning.

Summary of Findings to Date:

Data collection was completed May 31st 2019, and analyses addressing the main aims of the study are underway. However, we have already presented and published multiple findings using the NICHES data, as well as results from the parent cohort:

  • Schechter, Fuemmeler et al. (2018) published an article in the International Journal of Environmental Research and Public Health examining the impact of a statewide smoking ban in North Carolina on cotinine levels during pregnancy. Results indicated banning smoking in public spaces significantly reduced ETS, as measured by cotinine levels, for non-actively smoking pregnant women. This article was featured on several news outlets (i.e., WUNC, WRAL) and chosen as the NIEHS Extramural Paper of the Month.
  • Findings published in Frontiers in Cell and Developmental Biology looked at the relationship between diet during pregnancy and child neurodevelopmental outcomes as well as the role of differentially methylated regions in regulating genomically imprinted genes in these associations (House et al., 2018). Results showed that greater adherence to a Mediterranean diet during pregnancy was associated with numerous positive neurobehavioral outcomes including lower parent ratings of child depressive symptoms, decreased odds for atypical behaviors and behaviors associated with autism spectrum disorders. In addition, adherence to a Mediterranean diet was associated with methylation differences for imprinted control regions of PEG10/SGCE in the full sample as well as MEG3 and IGF2 in males.
  • Results published in Nicotine & Tobacco Research examined the interaction between prenatal cotinine and depressive symptoms during pregnancy in predicting birthweight (Schechter, Do et al., 2018). Results indicated that greater smoke exposure during pregnancy was associated with lower birthweight babies, but only in the context of greater depressive symptoms. Further, among women with the highest depressive symptoms, higher levels of cotinine was predictive of having a low birthweight baby (i.e., <2500 grams). In addition, African American women were found to have the lowest birthweight babies.
  • Dozmorov et al. (2018) published results in Brain, Behavior, & Immunity examining the relationship between maternal cytokine levels analyzed from the maternal prenatal blood samples and child cognitive functioning, as measured by the DAS-II and NIH Toolbox, collected as part of NICHES. Results showed that higher IL-12p70 and IL-17A levels were related to higher DAS-II General Conceptual Ability (GCA) scores, and higher IL-1β was related to lower GCA scores. Higher IL-12p70 was also associated with better performance on NIH toolbox measures of executive functioning related to inhibitory control, attention, and cognitive flexibility. Results indicated that dysregulation in immune activity during pregnancy is linked to later child cognitive ability and executive functioning.
  • Gao et al., (2018) published results in the journal Clinical Epigenetics indicating that prenatal tobacco smoke exposure was linked to greater methylation levels in the AXL gene body and that children who were exposed to smoke in utero had higher methylation levels in this region compared to unexposed subjects. Further, the combination of higher AXL methylation and prenatal tobacco smoke exposure elevated the risk of recent episodes of bronchitic symptoms in childhood.
  • Do et al (2019) published results in Pediatric Obesity looked at the relationship between methylation at differentially methylated regions (DMRs) of imprinted genes and children's Body Mass Index (BMI), as well as the mediating role of appetitive trains and birthweight and the moderating role of sex. Results did not indicate that appetitive traits and birthweight mediating the relationship between methylation at the DMRs. However, increased insulin‐like growth factor 2 DMR methylation was associated with greater satiety responsiveness, and that higher satiety responsiveness was associated with lower childhood BMI. Further, the associations between methylation at DMRs, appetitive traits, and BMI differed by sex. Results suggested that epigenetic profiles may mediate appetitive traits that can result in a greater likelihood for obesity.
  • Barry et al (2019) published an article discussing the development of an internally-created automated text-messaging platform designed to enhance retention and data collection within NICHES study as well as affiliated projects. Results reported in JMIR Public Health and Surveillance indicated that use of the platform was associated with greater participation in a study-related task and suggested that the platform could serve as a model for other systems used for enhancing communication and engagement with participants completing longitudinal studies.
  • Fuemmeler et al. (2019) published results examining the role of pre-pregnancy weight and gestational weight gain with ADHD symptoms and executive functioning in preschool children in the journal International Journal of Environmental Research and Public Health. Findings indicated that pre-pregnancy BMI was positively associated with higher ADHD symptoms and worse executive self-regulation behaviors. Less than adequate gestational weight gain was also related to more ADHD hyperactive-impulsive symptoms, whereas greater than adequate weight gain was related to more problematic behaviors associated with working memory and planning. The findings supported a relationship between maternal weight in pregnancy and offspring neurodevelopmental outcomes.
  • Results published in Epigenetics examined maternal pre-pregnancy obesity, offspring cord blood methylation, and childhood cardiometabolic health (Martin et al., 2019). Findings showed multiple differentially methylated CpG sites in umbilical cord blood among male and female offspring. In particular, CpGs in or near the TAPBP gene were highly associated with maternal obesity in both male and female offspring. Findings showed an association between pre-pregnancy maternal obesity-related CpG sites of TAPBP gene with children's cardiometabolic outcomes with sex specific effects.

Data has also been presented at several national and international conferences, including the Society for Research on Nicotine and Tobacco, the American Professional Society of ADHD and Related Disorders, American Society of Preventive Oncology, United States DOHaD Society meeting, NIDA Genetics Consortium, NIEHS FEST, and the NIEHS Children's Environmental Health Center's Meeting.

Several manuscripts describing results from Project 1 are currently under review and multiple papers are in preparation. In particular, with data collection complete and analyses underway, we expect publications addressing the primary aims to be submitted for publication within the year.

PROJECT 2: Mechanisms of Neurobehavioral Dysfunction from Developmental Nicotine & Tobacco

Investigators: Edward D. Levin, PhD, Theodore Slotkin, PhD, Frederic Seidler, PhD, Lisa Satterwhite, PhD

Project 2 has three major components, including an animal (rat) model of nicotine or tobacco smoke exposure prior to and throughout pregnancy, studies of neurochemistry in the brains of the exposed rats as well as in rat neural progenitor cells (primary and an established cell line) in vitro, and studies of tobacco smoke extract (TSE) exposure using hESCs that are differentiated into neural progenitors. The research was conducted with quality assurance procedures to insure reproducibility.

Aim 1. Determine gender-specific behavioral phenotypes resulting from developmental nicotine and ETS exposure.

Active maternal smoking has adverse effects on neurobehavioral development of the offspring, with nicotine providing much of the underlying causative mechanism. To determine whether the lower exposures caused by second-hand smoke are deleterious, we administered tobacco smoke extract (TSE) to pregnant rats starting preconception and continued through the second postnatal week, corresponding to all three trimesters of fetal brain development. Dosing was adjusted to produce maternal plasma nicotine concentrations encountered with second-hand smoke, an order of magnitude below those seen in active smokers. We then compared TSE effects to those of an equivalent dose of nicotine alone, and to a ten-fold higher nicotine dose. Gestational exposure to TSE and nicotine significantly disrupted cognitive and behavioral function in behavioral tests given during adolescence and adulthood (postnatal weeks 4-40), producing hyperactivity, working memory deficits, and impairments in emotional processing, even at the low exposure levels corresponding to second-hand smoke. Although TSE effects were highly correlated with those of nicotine, the effects of TSE were much larger than could be attributed to just the nicotine in the mixture. Indeed, TSE effects more closely resembled those of the ten-fold higher nicotine levels, but still exceeded their magnitude. In combination with our earlier findings, the present study thus completes the chain of causation to prove that second-hand smoke exposure causes neurodevelopmental deficits, originating in disruption of neurodifferentiation, leading to miswiring of neuronal circuits, and as shown here, culminating in behavioral dysfunction. As low level exposure to nicotine alone produced neurobehavioral teratology, "harm reduction" nicotine products do not abolish the potential for neurodevelopmental damage.

Tobacco exposure during development leads to neurobehavioral dysfunction in children, even when exposure is limited to secondhand smoke. We have previously shown in rats that developmental exposure to tobacco smoke extract (TSE), at levels mimicking secondhand smoke, starting preconception and extending throughout gestation, evoked subsequent locomotor hyperactivity and cognitive impairment. These effects were greater than those caused by equivalent exposures to nicotine alone, implying that other agents in tobacco smoke contributed to the adverse behavioral effects. In the present study, we examined the critical developmental windows of vulnerability for these effects, restricting TSE administration (0.2 mg/kg/day nicotine equivalent, or DMSO vehicle, delivered by subcutaneously-implanted pumps) to three distinct 10 day periods: the 10 days preceding mating, the first 10 days of gestation (early gestation), or the second 10 days of gestation (late gestation). The principal behavioral effects revealed a critical developmental window of vulnerability, as well as sex selectivity. Late gestational TSE exposure significantly increased errors in the initial training on the radial-arm maze in female offspring, whereas no effects were seen in males exposed during late gestation, or with either sex in the other exposure windows. In attentional testing with the visual signal detection test, male offspring exposed to TSE during early or late gestation showed hypervigilance during low-motivating conditions. These results demonstrate that gestational TSE exposure causes persistent behavioral effects that are dependent on the developmental window in which exposure occurs. The fact that effects were seen at TSE levels modeling secondhand smoke, emphasizes the need for decreasing involuntary tobacco smoke exposure, particularly during pregnancy.

Tobacco smoke is a complex mixture that includes thousands of compounds. Previously we have found that gestational exposure to the complex mixture of tobacco smoke extract caused long-term neurobehavioral impairments. In this study, we examined the interaction of two of the most biologically active, nicotine and benzo[a]pyrene (BaP). Developmental effects were determined in Sprague-Dawley rats prenatally exposed to low doses of BaP and nicotine (0.03 mg/kg/day of BaP and 2 mg/kg/day of nicotine) via maternal osmotic minipumps throughout gestation. Behavioral function was assessed in the offspring via a battery of tests through adolescence into adulthood. There were sex-selective effects in four of the behavioral tests. In the elevated plus maze, there was a significant interaction of BaP and sex, where BaP-treated males showed a trend for increased activity. In the novelty suppressed feeding test, there were significant sex selective effects in males such that the normal sex difference in the behavior in this test was eliminated. Male offspring with prenatal exposure to either nicotine or BaP showed significant reductions in fear response. In the Figure-8 locomotor activity test, BAP-exposed male offspring were significantly hyperactive. This also eliminated the sex difference normally seen in this test. This effect persisted into adulthood. In the attention task, males exposed to nicotine during gestation showed a significant percent hit impairment. BaP reversed this effect. No significant effects were seen with percent correct rejection. These data show that both nicotine and BaP cause persisting sex-selective behavioral effects that persist into adulthood.

Aim 2. Determine the deficits in specific neural circuits that cause the behavioral anomalies: In Vivo Neurochemistry.

These studies were designed to identify the synaptic pathways that underlie the long-term, behavioral effects of developmental exposure to TSE as compared to nicotine. We focused on acetylcholine (ACh) and serotonin (5HT) systems, pathways known to play major roles in the cognitive and emotional behaviors found to be targeted by these treatments, and known from previous studies to be specific targets for developmental exposure to nicotine. We simulated nicotine concentrations encountered with second-hand smoke, an order of magnitude below those seen in active smokers, and compared TSE to an equivalent dose of nicotine alone, and to a ten-fold higher nicotine dose, with exposures occurring throughout gestation and into the second postnatal week. We conducted longitudinal evaluations in multiple brain regions, starting in adolescence (postnatal day 30) and continued to full adulthood (day 150). TSE exposure impaired presynaptic ACh activity, exacerbated by a decrement in nicotinic nicotinic ACh receptor concentrations. Although both nicotine doses also produced presynaptic ACh deficits, these were partially compensated by hyperinnervation and receptor upregulation, effects that were absent with TSE. TSE also produced deficits in 5HT receptors that were not seen with nicotine. Regression analysis showed a profound sex difference in the degree to which nicotine could account for overall TSE effects: whereas the two nicotine doses accounted for 36-46% of TSE effects in males, it accounted for only 7-13% in females. Our results show that the adverse effects of TSE on neurodevelopment exceed those that can be attributed to just the nicotine present in the mixture, and further, that the sensitivity extends down to levels commensurate with second-hand smoke exposure. Since nicotine itself evoked deficits at low exposures, "harm reduction" nicotine products do not eliminate the potential for neurodevelopmental damage.

This was followed by a study to evaluate critical exposure periods for TSE, with three distinct, ten-day windows: premating, early gestation or late gestation. Just as in the continuous TSE exposure study, TSE in any of the three windows impaired presynaptic ACh activity, exacerbated by a decrement in nicotinic ACh receptor concentrations. Although the adverse effects were seen for all three treatment windows, there was a distinct progression, with lowest sensitivity for premating exposure and higher sensitivity for gestational exposures. 5HT receptors were also reduced by TSE exposure with the same profile: little effect with premating exposure, intermediate effect with early gestational exposure and large effect with late gestational exposure. As 5HT circuits can offset the neurobehavioral impact of cholinergic deficits, these receptor changes were maladaptive. Thus, there is no single "critical period" for effects of low-level tobacco smoke but there is differential sensitivity dependent upon the developmental stage at the time of exposure. Our findings reinforce the need to avoid secondhand smoke exposure not only during pregnancy, but also in the period prior to conception, or generally for women of childbearing age.

The third study focused on whether the greater effects of TSE as compared to nicotine reflected the polycyclic aromatic hydrocarbons present in TSE. We compared the developmental neurotoxicity of nicotine to that of the PAH archetype, benzo[a]pyrene (BaP), and also evaluated the effects of combined exposure to assess whether PAHs might exacerbate the adverse effects of nicotine. Nicotine or BaP alone impaired indices of ACh presynaptic activity, accompanied by upregulation of nicotinic ACh receptors and 5HT receptors. Combined treatment elicited a greater deficit in ACh presynaptic activity than that seen with either agent alone, and upregulation of nAChRs and 5HT receptors was impaired or absent. The individual effects of nicotine and BaP accounted for only 60% of the combination effects, which thus displayed unique properties. Importantly, the combined nicotine + BaP exposure recapitulated the effects of TSE, distinct from nicotine. Our results show that the effects of nicotine on development of ACh and 5HT systems are worsened by BaP coexposure, and that combination of the two agents contributes to the greater impact of TSE on the developing brain. These results have important implications for the relative safety in pregnancy of nicotine-containing products compared to combusted tobacco, both for active maternal smoking and secondhand exposure, and for the effects of such agents in "dirty" environments with high PAH coexposure.

Additional in vivo studies focused on the final stages of brain development, in adolescence. In this case, we were interested specifically in nicotine, given the decline in cigarette usage and rise in vaping among teenagers. Our key findings were that: (1) adolescent nicotine exposure reprograms the response of cerebrocortical 5HT systems to subsequent nicotine administration in adulthood, changes that may contribute to life-long vulnerability to relapse and re-addiction; and (2) for autonomic responses, adolescents are relatively resistant to withdrawal stress, which is likely to make episodic nicotine exposure less stressful or aversive than in adults. We also found long-term changes in cardiac norepinephrine after adolescent nicotine exposure, that resemble those known to be associated with risk of hypertension in young adulthood or subsequent congestive heart disease.

Aim 3. Determine cellular and molecular mechanisms for nicotine/tobacco extract-induced developmental neurotoxicity: In Vitro Models

We used two complementary models of neurodifferentiation: (1) Embryonic neural stem cells (NSCs), which can be used to explore the early decision to form neurons vs. glia; and (2) PC12 cells, which are already committed to the neuronal phenotype, and which make the subsequent decisions of neurotransmitter specification and neurite outgrowth. In the NSC model, nicotine diverted neurodifferentiation away from the glial phenotype while having little or no effect on the neuronal phenotype; TSE had this effect, too, but was primarily cytotoxic. On the other hand, there were much more profound effects in the PC12 model. TSE promoted the transition from cell replication to differentiation, resulting in fewer, but larger cells with greater neurite extension. TSE also biased differentiation into the dopaminergic versus the cholinergic phenotype, evidenced by an increase in tyrosine hydroxylase activity but not choline acetyltransferase. Nicotine likewise promoted differentiation at the expense of cell numbers, but its effect on growth and neurite extension was smaller than that of TSE; furthermore, nicotine did not promote the dopaminergic phenotype. Thus, as with the in vivo findings, the effects of TSE were much more profound than those of nicotine, and were directed toward neurodevelopmental stages akin to those in late gestation. We further were able to demonstrate the same synergistic effect of BaP with nicotine to recapitulate the effects of TSE in the PC12 model. This cements the mechanistic relationship of direct effects of TSE on neurodifferentiation, culminating in the abnormalities of brain circuitry and behavior observed in vivo.

Finally, we used the PC12 model to explore the possibility of ameliorating the effects of TSE with various treatment strategies: ACh receptor blockade, antioxidants, methyl donors. TSE acts through mechanisms beyond those of nicotine (not blocked by mecamylamine, a nicotinic receptor blocker); there are partial contributions from oxidative stress (approximately 50% protection from effects on cell replication, cell loss, cell growth) but relatively little contribution from effects on one-carbon metabolism (no protection from methyl donors). The methyl donors do, however, synergize with the antioxidants to provide nearly full protection against effects of TSE on cell growth. Notably, we found that none of the ameliorants protected the cells from effects on neurodifferentiation endpoints and in fact, the antioxidants themselves produced a substantial shift in the opposite direction, toward the cholinergic phenotype and away from the dopaminergic phenotype. Amelioration strategies may thus themselves be disruptive to neurodifferentiation while producing only modest protection against TSE. The effects of TSE clearly reside in multiple mechanisms rather than just a single effect, and it may prove quite difficult to come up with a strategy to prevent adverse effects.

Supplemental Funding was received from NIEHSfor Investigations of Sex Differences in Cognition. Sex differences in cognitive processing and function have been documented in human and animal studies. Females have been found to perform better than males on non-spatial memory tasks, while males tend to outperform females on spatial memory tasks. The neural mechanisms underlying these sexual dimorphisms are unclear. However, it is known that the cholinergic system is critically involved in memory processes, and there are notable differences between males and females in cholinergic system function and receptor expression. In particular, there are sex differences in the processing of information in the frontal cortex and hippocampus. In this study, we examined the roles of muscarinic and nicotinic acetylcholine receptors in the medial frontal cortex (MfC) and ventral hippocampus (VH) on spatial working memory in male and female rats. Local infusions of scopolamine (SCOP) and mecamylamine (MEC) (10, 20, 50 µg/side) were used to antagonize these receptors in each respective brain region during performance in the 16-arm radial arm maze. Infusions of SCOP into the VH caused a significant increase in memory errors in female rats, but had no significant effect on males, while infusions of MEC into the VH had no effect on either sex. Infusions of both SCOP (50 µg/side) and MEC (20 µg/side) into the MfC caused working memory impairments in both sexes. These results show that muscarinic acetylcholine receptors in the VH are differentially vulnerable to spatial memory impairments in females. Ventral hippocampal muscarinic acetylcholine receptors may play a key role in male-female differences in spatial memory.

NICHES Project 2 Satterwhite (Co-Investigator)

A human embryonic stem cell-derived human neural stem cell (hNSC) in vitro model was developed to identify mechanisms of nicotine and TSE toxicity in both male and female hNSCs. Rat neurotypic PC12 cells were analyzed for nicotine and TSE exposure phenotypes using new live cell methods that allowed direct measurements of cell number, viability, mitosis, apoptosis and geometry of "neurite" outgrowth in living PC12 cells for the first time. Before this, cell number, viability, "neurite" outgrowth had been measured indirectly using biochemical tests of mixed populations. Protrusions or "neurites" were longer and more branched following nicotine exposure compared to controls. The hNSC in vitro model was then developed which allows phenotyping of cell division and differentiation in the exact same cell cultures.

The initial studies used WA09-derived (female) hNSCs sourced from a commercial supplier. After optimizing growth and differentiation conditions, nicotine and TSE exposure phenotypes were scored in living cells for the first time. Significant findings include:

  • A 48 hour exposure to nicotine or TSE in proliferating cells elicits the same phenotypes as continual exposure into the differentiation phase.
  • Nicotine exposure inhibits cell division and reduces cell number; TSE exposure increases apoptosis.
  • The soluble vitamin C transporter 2 (SVCT2), which is the major ascorbic acid transporter in neural cells, is down-regulated by nicotine.
  • Co-administration of ascorbic acid with nicotine prevents down-regulation of SVCT2.

Approved lines WA01 (male) and WA09 (female) human embryonic stem cell lines were obtained from WiCell and differentiated into multiple isolates of neural stem cells. Additional studies included a survey of common nutritional supplements for the ability to remediate nicotine inhibition of cell proliferation and a study of cell fate in female compared to male hNSCs. DNA and RNA were archived from multiple exposure experiments. Rebecca Fry (UNC-CH Superfund Research Center) determined levels of heavy metals in the tobacco smoke extract used in the NICHES work indicating likely co-exposure of nicotine and numerous neurotoxic metals.

Two important insights emerged:

  • Given that 1) ascorbic acid can delay onset of Huntington's disease in the mouse model for Huntington's, 2) vitamin C promotes differentiation of dopaminergic neurons in the mouse midbrain, 3) vitamin C intact is associated with reduced risk for Parkinson's disease in humans, and 4) children taking vitamin C report reduced hyperactivity symptoms of ADHD, it is feasible to consider future studies to determine value of ascorbic acid therapy in children exposed to environmental nicotine.
  • Human neural stem cells are far more sensitive to nicotine and TSE than neurotypic PC12 cells. This suggests that neurotoxicity scored in rat cancer cells underestimates neurotoxicity to the human neural stem cell population.

Three planned publications include: 1) cell proliferation and differentiation phenotypes of nicotine and TSE exposure in human neural stem cells, 2) down-regulation of the vitamin C transporters by nicotine in hNSCs and 3) remediation of the growth and differentiation inhibition of nicotine by co-exposure with ascorbic acid in hNSCs.

PROJECT 3: Epigenomic Consequences of Early Life Environmental Tobacco Smoke Exposure

Investigators: Susan K. Murphy, PhD and David Corcoran, PhD

Maternal tobacco smoke exposure is associated with altered DNA methylation at genes implicated in autism spectrum disorder and attention deficit hyperactivity disorder in both rats and humans. Maternal tobacco smoke (TS) exposure is associated with adverse developmental outcomes in the exposed fetus, including low birth weight, preterm birth, and a host of neurological disorders such as attention-deficit hyperactivity disorder (ADHD) and autism spectrum disorder (ASD). These exposure-induced developmental outcomes have in part been attributed to changes in DNA methylation that occur during fetal development. Umbilical cord blood is commonly used as a proxy for exposure detection in humans during perinatal development. Others and we have previously associated maternal smoking with changes in DNA methylation at human metastable epialleles and imprinted loci in umbilical cord blood, suggesting the vulnerability of the epigenome to TS exposure during early development. Maternal smoking was also associated with changes in DNA methylation at genes involved in neurological function in the blood of exposed offspring. However, whether or not these changes correspond to similar DNA methylation changes in the brain is less understood. Here, we used whole genome bisulfite sequencing (WGBS) to identify genome-wide DNA methylation changes in the blood, cortex, and hippocampus of male rats that were developmentally exposed to tobacco-smoke extract (TSE), low dose nicotine (.2mg/kg/day) or high dose nicotine (2mg/kg/day) as compared to controls (Aim 1). We also used RNA Seq of the cortex and hippocampus of the same rats to identify potential changes in gene expression associated with TSE and/or nicotine exposure (Aim 2).

Using nearest gene name as a proxy for differentially methylated region (DMR), we identified 1,797 common DMRs across these three tissue types in TSE exposed rats, 2,153 common DMRs in low-dose nicotine exposed rats, and 2,010 common DMRs in high-dose nicotine exposed rats. In order to determine functional relevance, we retained DMRs showing any correlation with gene expression in either brain region and then clustered common DMRs by functional annotation clustering using the Database for Annotation, Visualization, and Integrated Discovery (DAVID). We identified genes involved in synapse formation as most highly enriched in each treatment group (Enrichment Score=ES; TSE ES=14.26, p=3.4e-15; 0.2 Nic ES=17.5, p=1.9e-21; 2.0 Nic ES=16.89, p=1.7e-17).

We next assessed genome scale DNA methylation in n=26 (13 high exposure, 13 low/no exposure) human cord blood samples using reduced-representation bisulfite sequencing (RRBS) in human male offspring exposed to maternal smoking, and identified 286 DMRs compared to males unexposed during pregnancy that were also common to TSE exposed rats. When comparing all data sets, we found an overlap of 110 DMRS common to rats of all three treatment groups and humans. Within this cluster, we identified 44 genes implicated in either autism spectrum disorder (ASD) and/or ADHD, a statistically significant enrichment (chi-square statistic 130.4; p<0.00001; comparing to the total number of ADHD/autism genes identified, 1,395, and the total number of protein coding genes in the genome, 21,306).

Working with Qiagen, we are beta testing a new platform that allows for targeted bisulfite sequencing of regions specified by the user, providing high throughput sequencing data for pooled indexed samples. We submitted all 44 regions of the identified ADHD/autism candidate genes identified for assay design to Qiagen; they provided the materials necessary for the library construction and are providing the sequencing. We are currently awaiting the sequencing results. We anticipate that this approach will provide validation of the identified targets using an independent method and in independent samples. These collective data will be compiled into a manuscript to be submitted in the next several months. Our findings are the first to suggest a relationship between exposure-induced DNA methylation changes in the blood and brain that are evident across species, and merit further investigation as a potential biomarker for exposure-induced neurological disorders.

To determine if there was a relationship between maternal gestational secondhand tobacco smoke exposure and gene methylation in the children (Aim 3) and in collaboration with Project 1, we made use of an existing dataset of HumanMethylation450 BeadChip data for a subset of our NEST cohort (n=79) for whom the mothers were themselves nonsmokers and had levels of prenatal cotinine <4 ng/ml, consistent with secondhand tobacco smoke exposure. Multivariable-adjusted linear regression models, adjusted for parity, race/ethnicity, mother's age at deliver, plate, batch and cell composition, yielded 11,575 cytosine-guanine dinucleotides (CpGs) sites that were differentially methylated at epigenome-wide statistical significance (False Discovery Rate ≤ 0.05), in relation to higher prenatal blood cotinine concentration. Functional enrichment analyses indicated that cotinine concentration levels were associated with altered DNA methylation of genes coding for proteins mapping to a number of metabolic and neurodevelopmental pathways.

As a follow-up to these analyses, we selected a separate random subsample of non-smoking women and performed bisulfite pyrosequencing for selected regions of genes, two of which were the most highly significant hits (AGER, n=139; PRKG1, n=142), one that has been repeatedly associated with smoke exposure (AHRR, n=115), and two others that were significant in meaningful functional enrichment pathways (TTC7B, n=115; CHAT1, n=114). Separate regression models using cotinine concentration levels in blood plasma to predict methylation values at each of the CpGs at these five genes, adjusted for by parity, mother's age at delivery, and race/ethnicity, were conducted. Higher cotinine concentration levels in our sample were associated with hypomethylation of the mean of three CpGs associated with AHRR (coefficient=0.75, p=0.02) and of position 1 associated with PRKG1 (coefficient=-1.07, p=0.01).

Results suggest that low levels of smoke exposure consistent with environmental smoke exposure are significantly related to changes in DNA methylation in regulatory regions of genes linked with active smoke exposure (AHRR) and genes related to neurodevelopment and self-regulation phenotypes (PRKG1).

We also generated DNA methylation data using bisulfite pyrosequencing for genes from the analysis of the WGBS and RRBS data described above. To address quality control for all pyrosequencing assays, we first performed validation of the assay design using defined mixtures of fully methylated and fully unmethylated bisulfite modified DNAs, purchased commercially from Qiagen. The assays are considered suitable for use on the biological specimens if there is an increase in the detection of DNA methylation for every incremental increase in the DNA methylation input into the reaction. We previously demonstrated that the sensitivity of bisulfite pyrosequencing is 0.5%. In unadjusted analyses using linear regression, we found that there are significant correlations between gestational cotinine levels measured in maternal blood and DNA methylation in the umbilical cord blood of their children for AHRR (n=180; R2=0.08; p<0.0002), CYP1A1 (n=178; R2>0.03; p<0.01), MEG3 (n=293; R2>0.01; p<0.05; strongest association in male offspring), MEG3-IG (n=278; R2=0.01; p<0.05) and HES1 (n=103; R2=0.06; p=0.02). We did not find significant correlations between DNA methylation and maternal cotinine levels for SLC6A2 (n=279), MMP9 (n=278), DRD4 (n=72) or NR3C1 (n=81).

Data are still being analyzed and a publication is planned describing these results. The central findings are that some but not all genes show shifts in DNA methylation in cord blood that are significantly correlated with maternal gestational cotinine measurements. Limitations of this approach are that the maternal cotinine measurements reflect a single time point in pregnancy, when the mothers were first recruited into the Newborn Epigenetics STudy. The levels of exposure to tobacco smoke may not be consistent throughout pregnancy. However, for the purposes of this study, we make the assumption that the mother's exposure to tobacco smoke at 13 weeks is reflective of her lifestyle.

Supplemental funding from NIEHS to study developmental nicotine exposure and masculinization of the rat preoptic area. Mammalian brain sexualization is developmentally mediated well after sex determination via gonadal hormones, and ultimately results in sexually dimorphic brain regions, both in structure and function. One such region, the preoptic area (POA), displays robust sexual dimorphism and is responsible for adult sexual behavior.

Estradiol-mediated masculinization of the POA occurs during early perinatal development in rats (E18-PN10) and triggers a downregulation of cell death pathways in males, resulting in larger POA volume in males compared to females. POA masculinization was also suggested to involve the inhibition of DNA methylation at sexually dimorphic differentially-methylated regions (DMRs), identified by whole genome bisulfite sequencing (WGBS), as well as the activation of methylation-dependent masculinizing genes (MDMGs) during postnatal days 0-4 in male rats. Although the mechanisms of POA sexualization are understood, little is known about how exposure to developmental toxicants may alter these pathways.

Tobacco use during pregnancy is common, and developmental nicotine exposure is known to impact DNA methylation. Further, a relationship between developmental nicotine exposure and altered sexual preference in exposed human females has been reported. Therefore, we hypothesized that developmental nicotine exposure would alter the sexualization of the POA in rats. We used a rat model of gestational nicotine exposure via osmotic minipump (2mg/kg/day nicotine) from premating through postnatal day 4 (PND4). A subset of PN4 POA was analyzed for MDMG expression using real-time PCR, as well as % methylation at sexually-dimorphic DMRs using bisulfite pyrosequencing. Males and females were gonadectomized at PN40 and fitted with testosterone capsules that mimic male circulating levels of testosterone. All animals were then paired with a hormonally stimulated female and assessed for male sexual behavior (#mounts, #intromissions, #ejaculations) at PN60. Following sex behavior assessment, POA area was measured using immunofluorescent staining techniques. Expression levels of individual MDMGs, including Cyp19a1, Ebf2, Ebf3, and Nr2f2, in PN4 POA did not display expected sex differences, however, across both sexes, treatment significantly varied in Cyp19a1 (1-way ANOVA, treatment main effect p=0.0326, p=0.0103 Student's t post hoc test between control and estradiol groups). Using a repeated measures ANOVA across all four MDMGs, relative expression across sexes was higher in nicotine-exposed PN4 POA compared to control animals (repeated measures ANOVA, p=0.0396). DNA methylation at the Nkain3 DMR was significantly lower in males and females of both the estradiol- and nicotine-treated groups, as well as in the control males, as compared to control females (2-way ANOVA, sex*treatment p=0.0097, p<0.05 Student's t post hoc test compared to control female).

Together, our findings suggest that developmental nicotine exposure is upregulating epigenetic mechanisms required for early POA masculinization. In adults, mount count in control males was higher than control females (p=0.0348). This sex difference was not distinguishable in either the estradiol- or nicotine-treated groups. Similarly, POA area was larger in control males versus control females (p=0.0133), but again this sex difference was not resolved in either the estradiol- or nicotine-treated groups. Mount count and POA area were normalized and combined in a repeated measures ANOVA to assess overall masculinization. Using this analysis, control females were significantly lower than all other groups (2-way ANOVA, sex*treatment p=0.0364, p<0.05 Student's t post hoc test compared to control female), supporting the masculinization of the nicotine-treated female brain. Our results further suggest that developmental nicotine exposure is capable of triggering the masculinization of the perinatal rat POA via epigenetic mechanisms. Further work is needed to determine how nicotine upregulates the epigenetic mechanisms contributing to brain masculinization, and whether or not developmental nicotine cessation could reverse these pathways.

Community Outreach and Translation Core: Community Education About Smoke Exposure (CEASE)

Investigator(s): Rochelle Schwartz-Bloom, Paul Bloom, Kathryn Pollak

In the first year of funding, we recruited a team of Duke students through the BASS Connections program at Duke to develop an educational science brochure on the effect of tobacco smoke exposure during pregnancy on the risk of causing ADHD in their children. The students developed the science brochure (in English, Spanish, and French) with oversight by the Project Leader and feedback from all Center Investigators. The brochure was also developed for health professionals, along with a poster to hang in health clinics. Finally, the student team developed an educational website (http://sites.duke.edu/helpbabiesavoidsmoke) that provided more detail about the educational information contained in the brochure.

Following the production of the science brochures, the students conducted a survey to assess how the educational information was received by women patients and their families in local OB/GYN clinics. Compared to a control brochure (published by the CDC), the CEASE brochure was significantly more effective in conveying new information about children's exposure to tobacco smoke and risk of ADHD (N = 241). Race/ethnicity did not contribute to the difference. Subjects with higher educational status scored significantly higher on the knowledge-based questions than subjects with lower education, regardless of brochure type. Last, language was a significant factor in knowledge, with English and French speakers scoring higher than Spanish speakers. We also obtained feedback from the health-care providers; 78% of them indicated that they were likely or very likely to share the brochure with their patients. All brochures that were left over from the study were disseminated to the public in various venues.

The CEASE brochures also contained an announcement to readers about an "Instagram contest". The contest asked the public to develop their own graphics/images describing what they've learned about environmental smoke exposure, ADHD, & epigenetics in the brochures, and on the "helpbabiesavoidsmoke" website. The contest was open for 2 months, and there were 7 winners. Two of the winning images were used in subsequent interventions.

Our team used two of the winning images to develop a public survey Instrument assessing the role of social media in education about tobacco smoke exposure and risk of ADHD in children. We conducted 2 field-trials with 1533 subjects. The intervention used Amazon M-Turk (an online survey instrument) to compare how the public would seek to get more information on about tobacco smoke exposure and risk of ADHD in children if they first saw our winning images embedded in Facebook, Twitter, and Instagram. As a control, we included our image/information on our website. We found that found that regardless of the format of the educational information (via website or 3 forms of social media), participants who were exposed to our educational material scored higher on intentions to reduce smoking, intentions to protect others from smoke, and knowledge based questions about the effect of tobacco smoke exposure during pregnancy on the risk of causing ADHD in their children. We also found that participants were significantly more likely to share the educational material when they saw it on our webpage versus seeing it on twitter, facebook, or instagram. However, we repeated the study with a smaller sample of 506 pregnant women. In this study, we found that pregnant women were very interested in the information regardless of how it was presented to them. Based on their responses, we concluded that they were significantly more motivated to engage in behaviors to protect babies from tobacco smoke. Therefore, we decided that pregnant women would be the best population to help inform the public about the effect of tobacco smoke exposure during pregnancy on the risk of causing ADHD in their children.

In our last study, we conducted a 12-week social media outreach activity on Facebook boosted to pregnant women or women interested in childbirth and nutrition, ages 18-45 in 6 different states (with low, medium, and high smoking rates). We compared still image posts vs video posts (from our educational materials developed previously) to determine which results in wider distribution. Our results reveal that we reached ~25,000-33,000 people per state with the still image posts, and ~4,200-7,300 people per state with the video posts (although at least 10,000 views were captured). It is not clear why Facebook had fewer views of video content vs image content. There was a significant difference in engagements (clicks, likes, shares, comments) with the posts; there were over 2000 viewers of the still images who engaged in the post, but none of the viewers of the video posts engaged. We concluded that it is not cost effective to use video to disseminate our information ($40/1000 people reached) compared to using still graphics ($7/1000 people reached).


Journal Articles: 48 Displayed | Download in RIS Format

Other center views: All 178 publications 54 publications in selected types All 48 journal articles
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Journal Article Breton CV, Marsit CJ, Faustman E, Nadeau K, Goodrich JM, Dolinoy DC, Herbstman J, Holland N, LaSalle JM, Schmidt R, Yousefi P, Perera F, Joubert BR, Wiemels J, Taylor M, Yang IV, Chen R, Hew KM, Freeland DM, Miller R, Murphy SK. Small-Magnitude Effect Sizes in Epigenetic End Points are Important in Children's Environmental Health Studies: The Children's Environmental Health and Disease Prevention Research Center's Epigenetics Working Group. Environmental Health Perspectives 2017;125(4):511-526. R835437 (Final)
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  • Journal Article Breton CV, Marsit CJ, Faustman E, Nadeau K, Goodrich JM, Dolinoy DC, Herbstman J, Holland N, LaSalle JM, Schmidt R, Yousefi P, Perera F, Joubert BR, Wiemels J, Taylor M, Yang IV, Chen R, Hew KM, Freeland DM, Miller R, Murphy SK. Small-magnitude effect sizes in epigenetic end points are important in children's environmental health studies:the Children's Environmental Health and Disease Prevention Research Center's Epigenetics Working Group. Environmental Health Perspectives 2017;125(4):511-526. R835437 (Final)
    R835436 (2017)
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  • Journal Article Breton CV, Marsit CJ, Faustman E, Nadeau K, Goodrich JM, Dolinoy DC, Herbstman J, Holland N, LaSalle JM, Schmidt R, Yousefi P, Perera F, Joubert BR, Wiemels J, Taylor M, Yang IV, Chen R, Hew KM, Hussey Freeland DM, Miller R, Murphy SK. Small-magnitude effect sizes in epigenetic end points are important in children’s environmental health studies: the Children’s Environmental Health and Disease Prevention Research Center’s Epigenetics Working Group. Environmental Health Perspectives 2017;125(4):511-526. R835437 (2017)
    R835432 (Final)
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  • Journal Article Small Magnitude Effect Sizes in Epigenetic Endpoints are Important in Children's Environmental Health Studies:The Children's Environmental Health and Disease Prevention Research Center's Epigenetics Working Group. Environmental Health Perspectives 2017; 125(4):511-526. R835437 (2016)
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  • Journal Article Cauley M, Hall BJ, Abreu-Villaca Y, Junaid S, White H, Kiany A, Slotkin TA, Levin ED. Critical developmental periods for effects of low-level tobacco smoke exposure on behavioral performance. Neurotoxicology 2018;68:81-87. R835437 (2017)
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  • Journal Article Fleisch AF, Kloog I, Luttmann-Gibson H, Gold DR, Oken E, Schwartz JD. Air pollution exposure and gestational diabetes mellitus among pregnant women in Massachusetts: a Cohort Study. Environmental Health 2016;15(1):40 (9 pp.). R835437 (Final)
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  • Journal Article Fleisch AF, Kloog I, Luttmann-Gibson H, Gold DR, Oken E, and Schwartz JD. Air Pollution Exposure and Gestational Diabetes Mellitus Among Pregnant Women in Massachusetts:a Cohort Study. Environmental Health 2016; 15:40-48. R835437 (2016)
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  • Journal Article Fuemmeler BF, Wang L, Iversen ES, Maguire R, Murphy SK, Hoyo C. Association between prepregnancy body mass index and gestational weight gain with size, tempo, and velocity of infant growth: analysis of the Newborn Epigenetic Study cohort. Childhood Obesity 2016;12(3):210-218. R835437 (2015)
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  • Journal Article Fuemmeler BF, Lee CT, Soubry A, Iversen ES, Huang Z, Murtha AP, Schildkraut JP, Jirtle RL, Murphy SK, Hoyo C. DNA methylation of regulatory regions of imprinted genes at birth and its relation to infant temperament. Genetics and Epigenetics 2016;8:59-67. R835437 (2017)
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  • Journal Article Gao L, Liu X, Millstein J, Siegmund KD, Dubeau L, Maguire RL, Zhang JJ, Fuemmeler BF, Kollins SH, Hoyo C, Murphy SK, Breton CV. Self-reported prenatal tobacco smoke exposure, AXL gene-body methylation, and childhood asthma phenotypes. Clinical Epigenetics 2018;10(1):98 (11 pp.). R835437 (2017)
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  • Journal Article Hall BJ, Cauley M, Burke D, Kiany A, Slotkin TA, Levin ED. Cognitive and behavioral impairments evoked by low-level exposure to tobacco smoke components: comparison with nicotine alone. Toxicological Sciences 2016;151(2):236-244. R835437 (2015)
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  • Journal Article Hall BJ, Abreu-Villaca Y, Cauley M, Junaid S, White H, Kiany A, Levin ED. The ventral hippocampal muscarinic cholinergic system plays a key role in sexual dimorphisms of spatial working memory in rats. Neuropharmacology 2017;117:106-113. R835437 (2017)
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  • Journal Article King KE, Kane JB, Scarbrough P, Hoyo C, Murphy SK. Neighborhood and family environment of expectant mothers may influence prenatal programming of adult cancer risk: discussion and an illustrative DNA methylation example. Biodemography and Social Biology 2016;62(1):87-104. R835437 (2015)
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  • Journal Article Lee W-C, Shen L, Catalano PJ, Mickley LJ, and Koutrakis P. Effects of Future Temperature Change on PM2.5 Infiltration in the Greater Boston Area. Atmospheric Environment 2017;150:98-105. R835437 (2016)
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  • Journal Article Lee W-C, Shen L, Catalano PJ, Mickley LJ, Koutrakis P. Effects of future temperature change on PM2.5 infiltration in the Greater Boston area. Atmospheric Environment 2017;150:98-105. R835437 (Final)
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  • Journal Article Levin ED. Learning about cognition risk with the radial-arm maze in the developmental neurotoxicology battery. Neurotoxicology and Teratology 2015;52(Pt A):88-92. R835437 (2014)
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  • Journal Article Murphy SK, Erginer E, Huang Z, Visco Z, Hoyo C. Genotype-epigenotype interaction at the IGF2 DMR. Genes 2015;6(3):777-789. R835437 (2014)
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  • Journal Article Nye MD, Fry RC, Hoyo C, Murphy SK. Investigating epigenetic effects of prenatal exposure to toxic metals in newborns: challenges and benefits. Medical Epigenetics 2014;2(1):53-59. R835437 (2013)
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  • Journal Article Nye MD, Hoyo C, Murphy SK. In vitro lead exposure changes DNA methylation and expression of IGF2 and PEG1/MEST. Toxicology In Vitro 2015;29(3):544-550. R835437 (2014)
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  • Journal Article Nye MD, King KE, Darrah TH, Maguire R, Jima DD, Huang Z, Mendez MA, Fry RC, Jirtle RL, Murphy SK, Hoyo C. Maternal blood lead concentrations, DNA methylation of MEG3 DMR regulating the DLK1/MEG3 imprinted domain and early growth in a multiethnic cohort. Environmental Epigenetics 2016;2(1):1-8. R835437 (2015)
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  • Journal Article Schechter JC, Kollins SH. Prenatal smoke exposure and ADHD: advancing the field. Pediatrics 2017;139(2):e20163481 (2 pp.). R835437 (2017)
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  • Journal Article Schechter JC, Fuemmeler, BF, Hoyo C, Murphy SK, Zhang JJ, Kollins SH. Impact of smoking ban on passive smoke exposure in pregnant non-smokers: using cotinine as a biomarker of exposure. International Journal of Environmental Research and Public Health 2018;15(1):E83 (16 pp.). R835437 (2017)
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  • Journal Article Slotkin TA, Card J, Seidler FJ. Adverse benzo[a]pyrene effects on neurodifferentiation are altered by other neurotoxicant coexposures: interactions with dexamethasone, chlorpyrifos, or nicotine in PC12 cells. Environmental Health Perspectives 2013;121(7):825-831. R835437 (2013)
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  • Journal Article Slotkin TA, Card J, Seidler FJ. Nicotine administration in adolescence reprograms the subsequent response to nicotine treatment and withdrawal in adulthood: sex-selective effects on cerebrocortical serotonergic function. Brain Research Bulletin 2014;102:1-8. R835437 (2014)
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  • Journal Article Slotkin TA, Card J, Stadler A, Levin ED, Seidler FJ. Effects of tobacco smoke on PC12 cell neurodifferentiation are distinct from those of nicotine or benzo[a]pyrene. Neurotoxicology and Teratology 2014;43:19-24. R835437 (2013)
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  • Journal Article Slotkin TA, Skavicus S, Card J, Levin ED, Seidler FJ. Amelioration strategies fail to prevent tobacco smoke effects on neurodifferentiation: nicotinic receptor blockade, antioxidants, methyl donors. Toxicology 2015;333:63-75. R835437 (2014)
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  • Journal Article Slotkin TA, Skavicus S, Card J, Stadler A, Levin ED, Seidler FJ. Developmental neurotoxicity of tobacco smoke directed toward cholinergic and serotonergic systems: more than just nicotine. Toxicological Sciences 2015;147(1):178-189. R835437 (2014)
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  • Journal Article Slotkin TA, Stadler A, Skavicus S, Seidler FJ. Adolescents and adults differ in the immediate and long-term impact of nicotine administration and withdrawal on cardiac norepinephrine. Brain Research Bulletin 2016;122:71-75. R835437 (2015)
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  • Journal Article Slotkin TA, Skavicus S, Card J, Levin ED, Seidler FJ. Diverse neurotoxicants target the differentiation of embryonic neural stem cells into neuronal and glial phenotypes. Toxicology 2016;372:42-51. R835437 (2016)
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  • Journal Article Slotkin TA, Stadler A, Skavicus S, Card J, Ruff J, Levin ED, Seidler FJ. Is there a critical period for the developmental neurotoxicity of low-level tobacco smoke exposure? Toxicological Sciences 2017;155(1):75-84. R835437 (2017)
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  • Journal Article Slotkin, T.A., Stadler, A., Skavicus, S., Card, J., Ruff, J., Levin, E.D., Seidler, F.J. 2016. Is there a critical period for the developmental neurotoxicity of low-level tobacco smoke exposure? Toxicological Sciences. DOW:10.1093. R835437 (2016)
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    Journal Article Smeester L, Yosim AE, Nye MD, Hoyo C, Murphy SK, Fry RC. Imprinted genes and the environment: links to the toxic metals arsenic, cadmium, lead and mercury. Genes 2014;5(2):477-496. R835437 (2014)
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  • Journal Article Soubry A, Hoyo C, Jirtle RL, Murphy SK. A paternal environmental legacy: evidence for epigenetic inheritance through the male germ line. BioEssays 2014;36(4):359-371. R835437 (2013)
    R835437 (2014)
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  • Journal Article Tindula G, Murphy SK, Grenier C, Huang Z, Huen K, Escudero-Fung M, Bradman A, Eskenazi B, Hoyo C, Holland N. DNA methylation of imprinted genes in Mexican-American newborn children with prenatal phthalate exposure. Epigenomics 2018;10(7):1011-1026. R835437 (2017)
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  • Journal Article Vidal AC, Benjamin Neelon SE, Liu Y, Tuli AM, Fuemmeler BF, Hoyo C, Murtha AP, Huang Z, Schildkraut J, Overcash F, Kurtzberg J, Jirtle RL, Iversen ES, Murphy SK. Maternal stress, preterm birth, and DNA methylation at imprint regulatory sequences in humans. Genetics and Epigenetics 2014;6:37-44. R835437 (2014)
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  • Journal Article Barry CM, Sabhlok A, Saba VC, Majors AD, Schechter JC, Levine EL, Streicher M, Bennett GG, Kollins SH, Fuemmeler BF. An Automated Text-Messaging Platform for Enhanced Retention and Data Collection in a Longitudinal Birth Cohort:Cohort Management Platform Analysis. JMIR Public Health Surveillance 2019 Apr 2;5(2):e11666. doi:10.2196/11666. PubMed PMID:30938689; PubMed Central PMCID:PMC6465978. R835437 (Final)
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  • Journal Article Dozmorov MG, Bilbo SD, Kollins SH, Zucker N, Do EK, Schechter JC, Zhang JJ, Murphy SK, Hoyo C, Fuemmeler BF. Associations between maternal cytokine levels during gestation and measures of child cognitive abilities and executive functioning. Brain Behavior, and Immunity 2018;70:390-397. doi:10.1016/j.bbi.2018.03.029. PubMed PMID:29588230; PubMed Central PMCID:PMC6471612. R835437 (Final)
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  • Journal Article Green AJ, Hoyo C, Mattingly CJ, Luo Y, Tzeng JY, Murphy SK, Buchwalter DB, Planchart A. Cadmium exposure increases the risk of juvenile obesity:a human and zebrafish comparative study. International Journal of Obesity (London) 2018 Jul;42(7):1285-1295. doi:10.1038/s41366-018-0036-y. PubMed PMID:29511319; PubMed Central PMCID:PMC6054604. R835437 (Final)
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  • Journal Article Slotkin TA, Skavicus S, Ko A, Levin ED, Seidler FJ. Corrigendum to "The Developmental Neurotoxicity of Tobacco Smoke Can Be Mimicked by a Combination of Nicotine and Benzo[a]pyrene:Effects on Cholinergic and Serotonergic Systems. Toxicological Sciences 2019 Mar 1;168(1):280. doi:10.1093/toxsci/kfy304. PubMed PMID:30615185. R835437 (Final)
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  • Journal Article Gonzalez-Nahm S, Mendez MA, Benjamin-Neelon SE, Murphy SK, Hogan VK, Rowley DL, Hoyo C. DNA methylation of imprinted genes at birth is associated with child weight status at birth, 1 year, and 3 years.Clinical Epigenetics 2018 Jun 28;10:90. doi:10.1186/s13148-018-0521-0. eCollection 2018. PubMed PMID:29988473; PubMed Central PMCID:PMC6025828. R835437 (Final)
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  • Journal Article Schechter J, Do EK, Zhang JJ, Hoyo C, Murphy SK, Kollins SH, Fuemmeler B. Effect of Prenatal Smoke Exposure on Birth Weight:The Moderating Role of Maternal Depressive Symptoms. Nicotine and Tobacco Research 2018 Dec 24. doi:10.1093/ntr/nty267. [Epub ahead of print] PubMed PMID:30590728. R835437 (Final)
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  • Journal Article Sorrow P, Maguire R, Murphy SK, Belcher SM, Hoyo C. Elevated metabolites of acetaminophen in cord blood of children with obesity. Pediatr Obes. 2019 Jan;14(1). doi:10.1111/ijpo.12465. Epub 2018 Sep 25. PubMed PMID:30253079. R835437 (Final)
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    Journal Article Hawkey A, Junaid S, Yao L, Spiera Z, White H, Cauley M, Levin ED. Gestational exposure to nicotine and/or benzo[a]pyrene causes long-lasting neurobehavioral consequences. Birth Defects Research 2019 Jul 31. doi:10.1002/bdr2.1568. [Epub ahead of print] PubMed PMID:31368242. R835437 (Final)
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  • Journal Article Martin CL, Jima D, Sharp GC, McCullough LE, Park SS, Gowdy KM, Skaar D, Cowley M, Maguire RL, Fuemmeler B, Collier D, Relton CL, Murphy SK, Hoyo C. Maternal pre-pregnancy obesity, offspring cord blood DNA methylation, and offspring cardiometabolic health in early childhood:an epigenome-wide association study. Epigenetics 2019 Apr;14(4):325-340. doi:10.1080/15592294.2019.1581594. Epub 2019 Mar 26. PubMed PMID:30773972; PubMed Central PMCID:PMC6557549. R835437 (Final)
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  • Journal Article Hawkey AB, White H, Pippen E, Greengrove E, Rezvani AH, Murphy SK, Levin ED. Paternal nicotine exposure in rats produces long-lasting neurobehavioral effects in the offspring. Neurotoxicology and Teratology. 2019 Jul-Aug;74:106808. doi:10.1016/j.ntt.2019.05.001. Epub 2019 May 16. PubMed PMID:31103693; PubMed Central PMCID:PMC6642846. R835437 (Final)
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  • Journal Article House JS, Mendez M, Maguire RL, Gonzalez-Nahm S, Huang Z, Daniels J, Murphy SK, Fuemmeler BF, Wright FA, Hoyo C. Periconceptional Maternal Mediterranean Diet Is Associated With Favorable Offspring Behaviors and Altered CpG Methylation of Imprinted Genes. Frontiers in Cell and Developmental Biology 2018 Sep 7;6:107. doi:10.3389/fcell.2018.00107. eCollection 2018. PubMed PMID:30246009; PubMed Central PMCID:PMC6137242. R835437 (Final)
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  • Journal Article Fuemmeler BF, Zucker N, Sheng Y, Sanchez CE, Maguire R, Murphy SK, Kollins SH, Hoyo C. Pre-Pregnancy Weight and Symptoms of Attention Deficit Hyperactivity Disorder and Executive Functioning Behaviors in Preschool Children. International Journal of Environmental Research and Public Health 2019 Feb;16(4):667-681. doi:10.3390/ijerph16040667. PubMed PMID:30823531; PubMed Central PMCID:PMC6406951. R835437 (Final)
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  • Journal Article Slotkin TA, Skavicus S, Ko A, Levin ED, Seidler FJ. The Developmental Neurotoxicity of Tobacco Smoke Can Be Mimicked by a Combination of Nicotine and Benzo[a]Pyrene:Effects on Cholinergic and Serotonergic Systems. Toxicological Sciences 2019 Jan 1;167(1):293-304. doi:10.1093/toxsci/kfy241. Erratum in:Toxicological Sciences 2019 Mar 1;168(1):280. PubMed PMID:30247698; PubMed Central PMCID:PMC6681680. R835437 (Final)
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  • Supplemental Keywords:

    Acetylcholine; ADHD; Attention deficit hyperactivity disorder; Benzo[a]pyrene; Brain sexualization; Cognitive function; Cortex; Cotinine; Depression; Developmental Origins of Health and Disease; Differentially methylated; Dissemination;l DNA methylation; DOHaD; Epigenetics; Genomic imprinting; Gestation; Hippocampus; Human neural stem cells; Hyperactivity; Intergenerational; In utero; Neural stem cells; Neurobehavior; Neurodifferentiation; Neurotransmission; Newborn Epigenetics STudy; Nicotine; NIH Toolbox; Public policy; Polycyclic aromatic hydrocarbons; Preoptic Area; Pyrosequencing; Rats; Reduced Representation Bisulfite Sequencing; RNA Sequencing; Secondhand smoke; Serotonin; Smoking ban; Social media; Sprague Dawley; Automated text messaging; Tobacco smoke exposure; Tobacco smoke extract Windows of exposure; Whole Genome Bisulfite Sequencing; Umbilical cord blood;

    Relevant Websites:

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    Progress and Final Reports:

    Original Abstract
  • 2013 Progress Report
  • 2014 Progress Report
  • 2015 Progress Report
  • 2016 Progress Report
  • 2017 Progress Report