2013 Progress 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. , Engelhardt, Barbara , Fuemmeler, Bernard , Hall, Brandon , Hoyo, Cathrine , Kollins, Scott H , Levin, Edward D , Satterwhite, Lisa , Seidler, Frederick , Slotkin, Theodore , Zhang, Weiwei
Current Investigators: Murphy, Susan K. , Fuemmeler, Bernard , Hoyo, Cathrine , Kollins, Scott H , Levin, Edward D , Satterwhite, Lisa , Schechter, Julia , Seidler, Frederick , Slotkin, Theodore , Wylie, Jamie
Institution: Duke University
EPA Project Officer: Nolt-Helms, Cynthia
Project Period: June 1, 2013 through May 31, 2018 (Extended to May 31, 2019)
Project Period Covered by this Report: June 1, 2013 through May 31,2014
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:

RD835437C001: Project 1 is examining 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 also is examining the relationship between smoke exposure, ADHD and DNA methylation. The objective of Project 1 is to evaluate the associations of both environmental tobacco smoke (ETS) exposure on cognitive and neurobehavioral outcomes across early development and examine the role of exposure-induced DNA methylation changes on these outcomes.

RD835437C002: Project 2 is determining 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 also is working 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. 

RD835437C003: Project 3 is investigating 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 are being applied to the 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 also will be examined in the in vitro models of neurodifferentiation and neurotransmission to determine associations with these phenotypes.

Community Outreach and Translation Core is developing 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. 

Specific aim 1: Develop a simple science primer that uses “lay language” to communicate the effects of ETS exposure (including nicotine) on the one’s genes (i.e., epigenetics). Similarly, communicate how these effects can be “handed down” to one’s offspring. Various stakeholders (e.g., doctors, pregnant patients, adolescent patients) will participate in the science primer development.

Specific aim 2: Disseminate the science primer in local community health centers to various stakeholders (e.g., doctors, pregnant patients, adolescent patients) along with a “call for contest applications” (see specific aim #4).

Specific aim 3: Develop a working document that translates the major findings of each of the three Center projects into “lay language” (add to the science primer in a section called “newest research”). Use the working document to develop the basis for a community-wide contest highlighting the basics about ETS and the newest research.

Specific aim 4: Hold a “contest.” Contestants (various stakeholders) use the science primer (from specific aim #2) along with the findings generated by the three Center projects (in specific aim #3) to construct their own YouTube videos and brochures that convey any of the NICHES findings. A committee of “judges” will choose a set of winning videos and brochures for a small field test and eventual dissemination.

Specific aim 5: Conduct a local field test in community and women’s health centers using the winning YouTube videos and brochures to determine the stakeholders’ attitudes and knowledge about the effects of ETS on themselves and their children.

Specific aim 6: Work with NIEHS to disseminate the YouTube videos and “science primers” within their database (and on the NIEHS website); post links to these materials online from the NICHES website and our science education website (www.rise.duke.edu), which has hundreds of thousands of hits.

Progress Summary:

RD835437C001: Project 1
 
The objective of Project 1 is to evaluate the associations of environmental tobacco smoke (ETS) exposure on cognitive and neurobehavioral outcomes across early development and examine the role of exposure-induced DNA methylation changes on these outcomes.
 
Aim 1: To characterize the extent and developmental timing of ETS exposure effects on cognitive and neurobehavioral outcomes in young children. This aim will be addressed once we have recruited sufficient participants for analysis.
 
Aim 2: To determine the relation between DNA methylation and cognitive and neurobehavioral outcomes in young children. This aim also relies on a larger number of individuals to be recruited into the study. Maternally Expressed Gene 3 (MEG3) methylation data is being generated in Project 3 for a subset of the potential participants.
 
We obtained human subjects approval from Duke, NIEHS and EPA and final approval of our Quality Management Plan (QMP) from the EPA in December 2013; human subjects research did not start until the QMP was approved.
 
Within the Newborn Epigenetic STudy (NEST) cohort (parent cohort), there are 759 eligible participants for NICHES Project 1. As of May 31, 2014, we have invited 394 of these cohort children to participate in the study. Of these 394, we have enrolled 19 children (68% African American, 32% Caucasian) with a mean age of 3.2 years. A total of 28 have refused participation and 347 are pending enrollment. Enrollment was slow during this period due to multiple factors, such as completing necessary training for staff, administering the assessments, staff turnover, a delay in funding due to the federal shutdown in 2013 and awaiting approvals for our QMP.
 
We have completed neurodevelopmental assessments of the 19 children and their mothers. For both mothers and their children, this includes assessments of executive functioning using the NIH toolbox and an IQ test. Three of these children provided blood and saliva samples, 3 children provided only blood sample and 13 children provided only saliva sample. These samples are being stored and will be used to assay cotinine and for studies of DNA methylation. Stored prenatal maternal blood will be assayed for cotinine and umbilical cord samples will be assayed for DNA methylation. Assays for cotinine from prenatal maternal blood are planned for completion in the coming year.
 
Our current battery of neurodevelopmental tests for the participants includes the NIH Toolbox with eight tasks; the Differential Abilities Scale (DAS) with four to six subtests; the Wechsler Abbreviated Scale of Intelligence–II (WASI) with four subtests; and parent self-report measures that include the Conners 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). During this report period, we have completed the full battery of tests on 18 mother and child dyads. One young child did not complete the assessment and will be rescheduled when the child is 4 years old.
 
RD835437C002: Project 2
 
In project 2, we are determining with rat models and cell lines the behavioral consequences of developmental exposure to tobacco smoke constituents and the neural mechanisms.
 
Aim 1. Determine the behavioral phenotypes resulting from developmental nicotine and ETS exposure.
 
In Vivo Behavior (Levin)
 
1. Exposed the first five cohorts of rats from preconception to the neonatal period via osmotic minipump to tobacco smoke extract (TSE) with a concentration delivering 0.2 mg/kg/day of nicotine modeling ETS, 0.2 mg/kg/day of nicotine alone, a higher dose of 2 mg/kg/day as a positive control modeling direct maternal smoking, and vehicle control. There are two litters administered each treatment in each cohort. One male and one female from each litter are tested in the behavioral assessment battery and one rat of each sex from each litter is sacrificed at each time point (birth, weaning, adolescence and adulthood) for neurochemistry and epigenetic analysis.
 
2. This study assessed the effects of nicotine and tobacco chemical mixture exposure during early development in Sprague-Dawley rats. Offspring of both sexes were assessed for locomotor hyperactivity, emotional dysfunction and cognitive impairment with the following test battery:
 
Week 4 Anxiety: Elevated Plus Maze
 
Week 5 Locomotor Activity: Figure-8 Maze
 
Week 6 Fear: Novel Environment Feeding
 
Week 7 Memory: Novel Object Recognition
 
Week 8-11: Radial-Arm Maze Learning
 
Week 12-40: Signal Detection Operant Attention Task
 
We have tested three cohorts on the behavioral test battery, including the figure-8 apparatus to assess locomotor hyperactivity, the elevated plus maze to assess anxiety, novel environment suppressed feeding to assess fear, novel object recognition to assess nonspatial memory, the 16-arm radial maze to assess spatial learning and memory, and the operant visual signal detection task to assess attention. The fourth cohort is in the midst of testing. The fifth cohort is being prepared for testing.
 
3. Preliminary behavioral results have thus far shown that gestational exposure to the TSE that delivers 0.2 mg/kg nicotine causes locomotor hyperactivity in the figure-8 apparatus that is significantly (p < 0.05) greater than groups treated with vehicle control solution and 0.2 mg/kg/day of nicotine alone, which was not seen to produce hyperactivity. The degree of hyperactivity produced by gestational exposure to TSE delivering 0.2 mg/kg/day was comparable to that produced by 2 mg/kg/day of nicotine alone. It is likely that other, non-nicotine constituent compounds present in tobacco potentiate the effect of nicotine such that gestational exposure to nicotine together with these other constituents in tobacco smoke produce hyperactivity in juvenile rats that is similar to that produced by nicotine alone at a 10-fold higher dose. The novel object recognition test shows that the TSE-exposed group has significantly less (p < 0.05) recognition of the novel object than controls. These results were presented at the 2014 Neurobehavioral Teratology Society meeting.
 
Aim 2. Determine the deficits in specific neural circuits that cause the behavioral anomalies.
 
In Vivo Neurochemistry (Slotkin and Seidler)
 
We have harvested and archived the seven brain regions for all treatment groups (control, TSE, nicotine 0.2 mg/kg/day, nicotine 2 mg/kg/day) at the four specified age points, ranging from adolescence through full adulthood, including generating the necessary samples for all the neurochemical determinations and epigenetics. We also developed a protocol that enables us to evaluate the necessary parameters for acetylcholine and serotonin systems in the same sample: choline acetyltransferase, hemicholinium-3 binding to the presynaptic choline transporter, nicotinic cholinergic receptor binding, 5HT1A and 5HT2 receptor binding. These analyses have now begun, and to date, we have made the measurements in four of the regions in adolescence. We are scheduled to complete these studies sometime this Spring. Our preliminary findings indicate that TSE, but not a comparable dose of nicotine, leads to downregulation of nAChRs and HC-3 binding; in contrast, nicotine increases ChAT relative to the values seen with TSE. These point to adverse effects of TSE on cholinergic systems over and above any contribution of nicotine.
 
Aim 3. Determine cellular and molecular mechanisms for nicotine/tobacco extract-induced developmental neurotoxicity.
 
In Vitro Studies
 
In vitro (Slotkin and Seidler): Our in vitro findings substantiate the same conclusion: TSE affects neurodifferentiation through mechanisms beyond just the contribution of nicotine. In our published study (Neurotoxicol. Teratol. 43:19-24, 2014), we showed that TSE promotes neurodifferentiation, resulting in a more rapid transition from cell replication to differentiation. Consequently, it produces deficits in the number of cells while promoting cell growth, neurite formation and emergence of neurotransmitter phenotypes; this effect was not seen with comparable concentrations of nicotine, although similar results could be elicited by raising the concentration of nicotine to 10x that found in TSE. TSE also diverted neurodifferentiation toward the dopaminergic phenotype at the expense of the cholinergic phenotype, effects that, if they occur in vivo, would disrupt the wiring of neural circuits. We are nearing completion of a second set of studies exploring the mechanisms by which TSE affects neurodifferentiation, focusing on three mechanistically based amelioration strategies: blockade of nicotinic receptors, antioxidants, and methyl donors (to offset any adverse effects on one-carbon metabolism). The latter two are notable in that these constitute some of the dietary recommendations made for pregnant smokers. Our findings, although not yet complete, confirm that 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.
 
In vitro (Satterwhite): To test the hypothesis that nicotine or TSE alters DNA methylation and differentiation, human neural stem cells (hNSCs) (Invitrogen) were exposed in vitro to 1 micromolar nicotine, TSE containing 1 micromolar nicotine (Arista) or vehicle control. DNA methylation was quantified by bisulfite pyrosequencing (Qiagen) for the ADHD-associated loci SCL6A2 (norepinephrine/dopamine transporter) and SLC6A3 (dopamine transporter). In preliminary experiments, DNA methylation was reduced at SLC6A2 in nicotine-exposed hNSCs relative to TSE or controls. In contrast, DNA methylation was reduced in SLC6A3 in both nicotine- or TSE-exposed hNSCs. During early differentiation, DNA methylation in SLC6A2 was increased in both nicotine- and TSE-exposed cells relative to controls but was unchanged at SLC6A3. DNA methylation at CHRNA4 (alpha subunit of the nicotinic acetylcholine receptor) increased ~4% in differentiating hNSCs but was unchanged by both treatments. These early results support that exposure to nicotine or TSE induces measurable changes in DNA methylation at genes highly relevant to ADHD.
 
RD835437C003: Project 3
 
We have generated preliminary data using cord blood specimens from the NEST, the parent study from which NICHES is recruiting. Preliminary data show an association between MEG3 and maternal smoking during gestation.
 
We have analyzed methylation levels at multiple candidate genes all of which have roles in neural function. We initially used a smaller subsample of cord blood specimens from smoke exposed versus non-exposed infants for screening purposes, and then developed methylation data for the promising candidates from the subsample analysis. We also performed experiments demonstrating that we are able to detect 0.5% changes in methylation by bisulfite pyrosequencing.
 
Aim 1: Identify ETS-related methylation targets. This is a future objective.
 
Aim 2: Identify ETS-altered methylation-expression relationships in frontal cortex. This is a future objective.
 
Aim 3: Determine if DNA methylation varies with ETS dose in humans. We obtained human subjects approval from Duke, NIEHS and EPA and final approval of our QMP from the EPA in December 2013. Human subjects research did not start until the QMP was approved.
 
We developed and validated pyrosequencing assays for the following candidate genes: MMP9, SLC6A2, FOSB (two regions) and CHRNA4. We tested methylation of these genes in a subset of prenatal smoke-exposed versus non-exposed infant umbilical cord blood (N = 15 in each group) to determine whether these genes would be worth pursuing in a larger dataset. Based on preliminary results, we moved forward with MMP9 and SLC6A2. We performed bisulfite pyrosequencing of umbilical cord blood specimens for MMP9 (N = 410; 239 non-exposed and 171 exposed) and SLC6A2 (N = 409; 240 non-exposed; 166 exposed). Analysis of the raw, unadjusted data indicated neither showed significant differences between infants exposed versus unexposed to tobacco smoke during gestation (p = 0.39 and p = 0.11, respectively). We are continuing analysis of these data with regard to potential confounding factors and will assess the results again once we have obtained plasma cotinine levels for the mothers during pregnancy.
 
https://cfpub.epa.gov/ncer_abstracts/images/fckimages/index.cfm?imgid=7991Text Box: % Methylation
 
We used a previously developed pyrosequencing assay for the differentially methylated region at the promoter of MEG3, a gene encoding a long, noncoding RNA that is highly expressed during human fetal development and of interest to neurobehavioral outcomes in Project 1. We analyzed 490 individuals and in unadjusted analyses, mothers who reported active smoking during pregnancy (N = 27) had infants with significantly higher methylation than those from mothers who reported never smoking and no other secondhand exposures (N = 226; p = 0.048).
 
We have designed pyrosequencing assays for human ADRA2A, DRD2, MAOA, NGF, OPRD1 and BDNF; optimization and validation of these assays is in progress.
 
https://cfpub.epa.gov/ncer_abstracts/images/fckimages/index.cfm?imgid=7993
 
In tests in which we wished to determine the sensitivity of pyrosequencing at low levels of methylation (0%-5% methylation), we prepared mixtures of unmethylated and methylated DNAs at 0.5% increasing increments of methylation. We indeed found that pyrosequencing was able to detect these small differences. To our knowledge, this is the first demonstration of the ability of pyrosequencing to detect 0.5% differences in methylation and at overall methylation levels previously considered to be background noise for this technology (i.e., less than 5% methylation).
 
We will be analyzing DNA methylation in the tissues derived from rats in project 2. We have designed Sequenom EpiTYPER assays to screen candidate genes, including TH, Dbh, Slc6a2, Slc18a1, Slc18a2, Adra2a and Adra2c in rat cerebellum from Dr. Slotkin’s work for areas of differential methylation in response to prenatal nicotine exposure (6 mg/kg/day). Once identified, pyrosequencing will be used to analyze these regions in cerebellum collected longitudinally every other day from prenatal day 4 through postnatal day 21 (synaptogenesis). This will help determine if these genes undergo dynamic changes in methylation during brain development and may help pinpoint the best timing for subsequent analyses in this project.
 
 Community Outreach and Translation Core
 
(Only specific aims 1-3 apply to the past funding period)
 
Development of the science primer (brochure): A team of four undergraduate students as part of the BASS Connections program at Duke were chosen to participate in the development of the CEASE materials and research implementation. During the first year, they helped to develop the CEASE brochure with oversight by the Project Leader and feedback from all Center Investigators. The students met with all Center Investigators to learn the background of the content that would be incorporated into the educational brochure. The students developed the science brochure in both English and Spanish. They developed a similar version for health providers who will be in the clinics for dissemination and a poster to hang on the walls of the clinic exam rooms. On the brochures and the poster, the “contest” is announced to the public (see below). The brochures are in the process of being printed (October 2014).
 
Development of a survey to be taken by stakeholders: To determine the impact of the brochure on clinic patients’ (and others’) attitudes and knowledge, the students created a survey to be taken online (via Qualtrics) at the clinics. The survey is also in Spanish. They devised a randomized methodology to deliver a “control brochure,” which is publically available from the Centers for Disease Control and Prevention (CDC) on the Web. They translated the control brochure into Spanish as well. This initial field test is an addition to the original specific aims, as the students felt is important to get feedback from the public about the educational brochure. All Center Investigators agreed. The field test will be implemented in the clinics during late fall 2014.
 
Development of the CEASE website to announce the contest: The student team developed the website called “Help Babies Avoid Smoke” (http://sites.duke.edu/helpbabiesavoidsmoke). This website announces the public contest to develop “Instagrams” depicting what they’ have learned from the brochures and the website itself (see below). Students incorporated the content from the brochures to be distributed in the clinics and from their meetings with the Center Investigators into a section called “Research.” They explain the key elements of the Center projects and how the information helps the public understand the impact of tobacco smoke exposure on child brain development, especially ADHD.
 
Development of the “contest”: Students felt that the initial proposal to develop YouTube videos for the contest would not allow enough of the public to participate and would be too difficult for many people. Instead, they suggested making the contest for Instagrams, in which the public could submit photos, graphics, or original artwork depicting what they have learned from the brochure and the website. With help from the Duke lawyers, they articulated the contest rules and guidelines for submission to the contest judges. They created the Help Babies Avoid Smoke website to announce the contest and provide additional educational information for submitting an Instagram (see above). The contest will be open as soon as the first clinic visit for the field test is made (late October 2014).
 
Recruitment of Durham Health Clinics: We obtained approval from the Durham County Health Department (3 clinics) and Durham Obstetrics & Gynecology (2 clinics) to visit their clinics and approach their patients and others in the waiting rooms to deliver the brochures and the field test (surveys online). Field-testing should begin late October 2014).

Future Activities:

RD835437C001: Project 1
 
Our recruitment started slowly, but we are making a rapid increase in recruitment and expect to be on target to achieve the aims. We have applied for funding through the National Institutes of Health (NIH) to further study executive functioning development, including eating behaviors. We are proposing to expand enrollment and further study how prenatal obesity might contribute to neurodevelopmental outcomes. It is possible that the contribution of both secondhand smoke exposure and prepregnancy obesity may further exacerbate neurodevelopmental outcomes.
 
RD835437C002: Project 2
 
Levin Lab
  • Finish neurobehavioral assessment for all cohorts of attention, learning, spatial working and reference memory, nonspatial memory, fear and anxiety for male and female rats exposed throughout early development to tobacco smoke extract (TSE) at a dose modeling environmental tobacco smoke exposure and the equivalent dose of nicotine (0.2 mg/kg/day) without the other constituents of TSE in comparison with vehicle control and a positive comparison group of a higher dose of nicotine (2 mg/kg/day), modeling the nicotine exposure from primary smoking. Determine the most sensitive neurobehavioral effects.
  • Investigate effects of briefer exposures to determine specific time windows of development (preconception, early prenatal, late prenatal or neonatal) that are most important for causing neurobehavioral dysfunction.
  • Begin assessments of pharmacotherapies to attenuate the neurobehavioral dysfunction caused by TSE and nicotine exposure during development.
Slotkin Lab
  • Perform brain regional dissections in an age range from birth through weaning, adolescence and adulthood of male and female rats exposed throughout development to TSE at a dose modeling environmental tobacco smoke exposure and the equivalent dose of nicotine (0.2 mg/kg/day) without the other constituents of TSE in comparison with vehicle control and a positive comparison group of a higher dose of nicotine (2 mg/kg/day), modeling the nicotine exposure from primary smoking.
  • Perform brain region-specific neurochemical analyses characterizing monoaminergic and cholinergic neurotransmitter systems of in vivo tissues from perinatal TSE administration.
  • Elucidate mechanisms underlying the TSE effects on neural cell replication, neurite outgrowth and differentiation in the PC12 model.
  • Prepare TSE-exposed samples from PC12 studies for epigenetics evaluations.
Satterwhite Lab
  • Characterize nicotine and TSE exposure phenotypes in human and rat neural stem cells (NSCs) by measures of EdU (S-phase), annexin V (apoptosis), propidium iodide (cell nonviability) and DAPI (cell number and mitotic index). Differentiation will be quantified by immunofluorescence and qPCR to identify neurons (MAP2, DCX), astrocytes (GFAP), oligodendrocytes (GALC), synaptic phenotypes (TH, ACh, AChR) and the undifferentiated state (NES); test the hypothesis that pretreatment with methyl donor micronutrients and antioxidants will rescue nicotine/TSE-associated exposure phenotypes. RNA and DNA will be isolated and archived for gene expression (Affymetrix human U133 Plus2.0 whole genome microarrays) and DNA methylation (Illumina Infinium HumanMethylation450BeadChip arrays) studies.
  • Concordance between DNA methylation patterns in maternal/child whole blood (Project 1) and in human neural stem cells (hNSCs) exposed to nicotine/TSE will be determined in select loci and genome/epigenome-wide studies. We will assay individual loci associated with prenatal exposure to nicotine and ADHD (Project 3) and loci associated with the developmental dopaminergic specification pathway. including PITX3 (paired like homeobox domain 3), NR4A2 (Nurr1), SLC6A2 (norepinephrine transporter), SLC6A3 (dopamine transporter 1), DRD1-5 (dopamine receptors 1-5) and EN1 (engrailed homeobox 1).
RD835437C003: Project 3
  • Identify targets of methylation in exposed humans and rats. We will progress with pyrosequencing and data analysis, adding additional genes for analysis, and with Sequenom screening to identify target regions in both humans and in rat tissues, as well as in cultured cells.
  • Once tissues from exposed/unexposed and behaviorally characterized rats are available from Project 2, we will generate RNA and DNA; pooled RNA will be used for whole transcriptome profiling to determine how gene expression in frontal cortex is altered by tobacco smoke and nicotine exposures, and the pooled DNA will be used for whole genome bisulfite sequencing to determine how the methylome is altered, to be carried out using funds from years 2 and 3 (due to expense).
  • The RNA from individual rat brain, liver and kidney tissues, as well as cultured cells, will be used to validate the expression levels detected by high-throughput transcriptome sequencing.
  • Cotinine assays will be run from maternal blood for the individuals undergoing analysis in Project 3 to enable analysis of the methylation data with respect to a continuous marker of exposure.
  • A human subjects protocol will be prepared for the North Carolina State Department of Health and Human Services Clinical Chemistry Laboratory to allow for analysis of lead levels in year 5.
  • We will continue to bank and track biological specimens as they are collected from Projects 1 and 2.
Community Outreach and Translation Core 
 
In the next year, we will carry out the first field test of the brochures in the participating clinics. Students will implement the online surveys in the clinics. Second, the Instagram contest will be conducted, with a finish date of December or January 1, so that the judging can take place. The second field-test (of the winning Instagrams) will take place in the spring of 2015 in the participating clinics. Once the second field test is completed, the students will conduct a survey of the health-care providers in the participating clinics. Analysis of results of both field tests and the clinic provider surveys will take place over the next year.


Journal Articles: 33 Displayed | Download in RIS Format

Other center views: All 116 publications 38 publications in selected types All 33 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, 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)
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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 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 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)
    R835437 (2017)
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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)
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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 Lee, W.-C., Shen, L., Catalano, P.J., Mickley, L.J., and Koutrakis, P. (2017). Effects of Future Temperature Change on PM2.5 Infiltration in the Greater Boston Area. Atmospheric Environment 150, 98-105. R835437 (2016)
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    Journal Article Carrie V. Breton, Carmen J. Marsit, Elaine Faustman, Kari Nadeau, Jaclyn M. Goodrich, Dana C. Dolinoy, Julie Herbstman, Nina Holland, Janine M. LaSalle, Rebecca Schmidt, Paul Yousefi, Frederica Perera, Bonnie R. Joubert, Joseph Wiemels, Michele Taylor, Ivana V. Yang, Rui Chen, Kinjal M. Hew, Deborah M. Hussey Freeland, Rachel Miller, and Susan K. Murphy. 2017. 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 (2016)
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    Journal Article Levin ED and Y Abreu-Villaça. Developmental neurotoxicity of nicotine and tobacco. In Handbook of Developmental Neurotoxicology, (C. Wang, M. Paule, and W. Slikker, Jr. eds.), Academic Press, San Diego, 2017. R835437 (2016)
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    Journal Article Fleisch, A.F., Kloog, I., Luttmann-Gibson, H., Gold, D.R., Oken, E., and Schwartz, J.D. (2016). Air Pollution Exposure and Gestational Diabetes Mellitus Among Pregnant Women in Massachusetts:a Cohort Study. Environmental Health 15, 1-9. R835437 (2016)
    R834798C005 (Final)
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    Journal Article Breton, C.V., Marsit, C.J., Faustman, E., Nadeau, K., Goodrich, J.M., Dolinoy, D.C., Herbstman, J., Holland, N., LaSalle, J.M., Schmidt, R., Yousefi, P., Perera, F., Joubert, B.R., Wiemels, J., Taylor, M., Yang, I.V., Chen, R., Hew, K.M., Hussey Freeland, D.M.,Miller, R. and S.K. Murphy. 2016. 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 (in press) DOI:10.1289/EHP595. R835437 (2016)
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    Supplemental Keywords:

    secondhand smoke, nicotine, tobacco smoke extract, attention deficit hyperactivity disorder, neurobehavior, cognitive function, neurotransmission, human neural stem cells, pyrosequencing, DNA methylation, epigenetics, Instagram

    Relevant Websites:

    http://niches.duke.edu Exit

    http://www.rise.duke.edu Exit

    http://sites.duke.edu/helpbabiesavoidsmoke Exit

    Progress and Final Reports:

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