2015 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.
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, 2015 through May 31,2016
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 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.

Our objective is to evaluate the associations of both environmental tobacco smoke (ETS) exposure on cognitive and neurobehavioral outcomes across early development and to 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. MEG3 methylation data is being generated in Project 3 for a subset of the potential participants.

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 is also 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.

Aim 1: Determine the behavioral phenotypes resulting from developmental nicotine and ETS exposure.

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

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

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 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.

Aim 1: Identify ETS-related methylation targets.

Aim 2: Identify ETS-altered methylation-expression relationships in frontal cortex.

Aim 3: Determine if DNA methylation varies with ETS dose in humans.

Progress Summary:

Project 1

During our third year for this study, we made significant progress in recruitment. Since beginning recruitment, there have been approximately 969 potentially eligible participants in our cohort that meet our inclusion criteria. As of March 29, 2016, we have enrolled 236 participants: 58.9% African American, 36.9% Caucasian, 2.1% multi-racial, 1.3% Asian, and 0.8% unknown race. The mean age is 4.3 years (sd 1.03; range 3-6). Ninety-six participants have refused or moved out of our catchment area and 186 have aged-out. The number who currently remain eligible are 451 women and their children over the age of four. We are actively reaching out to these participants through mail, recruitment events, or meeting them in clinics in order to enroll them for NICHES.

We have completed neurodevelopmental assessments of the 236 children and their mothers at time 1, and 2 mother-child dyads have completed the 2-year follow-up visit. For both mothers and their children, this includes assessments of executive functioning using the NIH toolbox and an IQ test. Among these children, we have collected saliva from nearly every participant (223 individual saliva samples at time 1 and 2 saliva samples at time 2), which will provide a method for assessing cotinine. Blood samples continue to be challenging to collect from young children, and we have recently started to offer the option of collecting blood via capillary tubes in addition to venipuncture. Overall, we have collected blood on 42 participants (39 via venipuncture and 3 via capillary) at time 1; we were unable to collect blood on either of the time 2 participants. All samples are being stored and will be used to assay cotinine and for DNA methylation. Stored prenatal maternal blood was recently assayed for cotinine and umbilical cord samples are being assayed for DNA methylation in selected differentially methylated regions of interest. Loci chosen are being informed through putative pathways, recent findings in the literature and findings from Project 2 and 3.

Our current battery of neurodevelopmental tests for the participants include the NIH Toolbox with 8 tasks, the Differential Abilities Scale (DAS) with 4-6 subtests, Wechsler Abbreviated Scale of Intelligence –II (WASI) with 4 subtests, parent self-report measures that include: 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). During this report period we have completed the full battery of tests on the entire 236 mother and child dyads who completed study visits.

Data analyses have included descriptive and summary statistics of cotinine levels. We are building multivariate models to assess associations between cotinine and neurodevelopmental outcomes.

With internal funding, Dr. Fuemmeler has led the development and implementation of a unique platform for increasing recruitment, retention and maintaining engagement of study participants. The platform uses a combination of communication channels [web, email, SMS text, interactive voice recording (IVR)] to facilitate interactive communication with cohort participants over the course of the study. The platform is programmed to semi-automatically perform many of the recruitment/retention procedures traditionally done by research assistants. For instance, the platform reads dates in our database and sends automated pre-programmed messages that we developed for delivery on specific dates and times (e.g., in advance of a study visit, child’s birthday,) or it sends generic messages to all participants on specific dates (e.g., quarterly requests to mailing address updates, links to a website). Messages are stored, so if the participant misses a call, they can call back at their convenience to receive the message. In addition, the interactive system is currently being used to collect data on specific behaviors, including sleep duration and quality by prompting participants to respond to simple queries (e.g., “using your keypad, enter the time your child went to bed”; “how many times did your child wake up?”) and remind participants to complete study related activities. For our ongoing studies, all procedures using this platform have been vetted through our information security office and have been approved by the IRB. We believe this method will enhance our ability to keep participants engaged throughout the study as well as in possible follow-up studies. The platform has launched and we are recruiting eligible NICHES participants to enroll in the system when they participate in other NEST studies.

In addition to digital methods we are using to enhance retention, we also have been sending newsletters twice yearly, thank-you cards, and holiday greeting cards.

Significance: This study will add data on the effects of second hand smoke exposure on child cognitive development and identify potential epigenetic signatures mediating these associations.

Project 2

This project 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 is also working to define the most sensitive developmental window(s) of vulnerability to tobacco smoke and nicotine exposure, which components of tobacco smoke are driving the effects and will determine if dietary interventions can ameliorate the effects of these exposures.

Aim 1: Determine the behavioral phenotypes resulting from developmental nicotine and environmental tobacco smoke (ETS) exposure.

In Vivo Behavior

We completed and published the behavioral results from the first of three major studies, which compared the long-term behavioral effects of gestational tobacco smoke extract (TSE) modeling environmental tobacco smoke exposure (second hand smoke) with the same amount of nicotine alone (0.2 mg/kg/day) or ten times that nicotine level (2 mg/kg/day) modeling the nicotine exposure of primary smoking (Hall, et al., 2016). The offspring were tested for persisting behavioral on the following test battery.

Age Test
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

The offspring of the rats exposed to low level TSE showed significant locomotor hyperactivity during adolescence 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. Hyperactivity was seen during adolescence but not in adulthood, which matches the clinical experience with ADHD in which the hyperactivity prominent during childhood and adolescence tends to diminish in ADHD residual type in adulthood. 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 ten-fold higher dose. The novel object recognition test showed that the TSE exposed group has significantly less (p<0.05) recognition of the novel object than controls. Both nicotine doses caused significantly reduced novel object recognition, effects though which were not as pronounced as was seen with TSE exposure. Thus, the low motivation novel object recognition test was very sensitive to the cognitive effects of gestational tobacco and nicotine exposure. Interestingly, the high motivation food deprivation motivated visual recognition task did not show a TSE exposure-induced impairment. Rather TSE exposure caused a slight though significant improvement in this task. Gestational nicotine exposure was not found to have a significant effect on this test. Apparently low motivation tests are more sensitive to the persistent deficits caused by gestational TSE exposure. Again, this resembles the cognitive impairment in ADHD. Cognitive performance in people with ADHD can be quite proficient under highly motivating circumstances, whereas it lapses under low motivating situations. This may be why stimulants are efficacious in ADHD therapy. They provide a degree of pharmacological motivation. In the next study we will in the same task test cognitive performance under high and low motivation circumstances. This study has been published in Toxicological Sciences.

We are nearly done with the second major study of the project, the critical windows of exposure study. We used iPrecio programmable pumps to provide the windows of exposure: ten days before mating, ten days during the first half of gestation and ten days during the second half of gestation. We have found that the late gestational TSE exposure caused significant locomotor hyperactivity during adolescence. This effect was less pervasive than that caused by TSE exposure from pre-mating through all of gestation in the first study. With only late gestational exposure the significant hyperactivity was seen during the early part of the hour-long session but not later when all the groups showed habituation to the environment and lower activity. In contrast, with the more complete TSE exposure from pre-mating until birth, locomotor hyperactivity was seen as a significant main effect across the entire locomotor activity test session. Late gestational TSE exposure was also found to cause significant anxiety like response on the elevated plus maze. The rats exposed to Low level TSE during the second half of gestation had significantly less time in the open arms of the elevated plus maze than controls.

We are preparing to begin the third major study of this grant period in which rescue and therapeutic treatments will be tested. The rescue treatment consists of antioxidants and methyl donors administered at the time of TSE exposure from pre-mating until birth. The therapeutic treatments will be evaluated during locomotor activity testing and cognitive assessment to see how effective they are in reducing TSE induced hyperactivity and cognitive impairment. The drug treatments to be tested are methylphenidate, amphetamine and atomoxetine. In addition we are conducting a study of the interaction of two of the principal bad actors in tobacco smoke, nicotine and the prototypic polyaromatic hydrocarbon, benzo-a-pyrene.

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

Parallel to the behavioral studies of TSE effects, we evaluated the developmental neurotoxicity TSE administered to pregnant rats starting preconception and continued through the second postnatal week. 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. 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 cholinergic activity, exacerbated by a decrement in nicotinic cholinergic receptor concentrations. Although both nicotine doses also produced presynaptic cholinergic deficits, these were partially compensated by hyperinnervation and receptor upregulation, effects that were absent with TSE. TSE also produced deficits in 5HT receptors in females 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. The consonance of these results with the behavioral findings thus provide a mechanistic underpinning for the adverse effects of TSE.

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

In our previous report, we detailed results with the PC12 cell model that found the same overall conclusion to our in vivo findings reported above: TSE has adverse neurodevelopmental effects that exceed those attributable to nicotine alone, producing outcomes more similar to 10-fold higher nicotine concentrations than those present in the extract. In the current year, we again used the PC12 model to evaluate the mechanisms by which TSE affects neurodifferentiation, and to examine whether ameliorative strategies directed at specific mechanisms could offset the effects of TSE. In undifferentiated cells, TSE impaired DNA synthesis and cell numbers to a much greater extent than nicotine alone; TSE also impaired cell viability to a small extent. In differentiating cells, TSE enhanced cell growth at the expense of cell numbers and promoted emergence of the dopaminergic phenotype. Nicotinic receptor blockade with mecamylamine was ineffective in preventing the adverse effects of TSE and actually enhanced the effect of TSE on the dopamine phenotype. A mixture of antioxidants (Vitamin C, Vitamin E, N-acetyl-L-cysteine) provided partial protection against cell loss but also promoted loss of the cholinergic phenotype in response to TSE. Notably, the antioxidants themselves altered neurodifferentiation, reducing cell numbers and promoting the cholinergic phenotype at the expense of the dopaminergic phenotype, an effect that was most prominent for N-acetyl-L-cysteine. Treatment with methyl donors (Vitamin B12, folic acid, choline) had no protectant effect and actually enhanced the cell loss evoked by TSE; they did have a minor, synergistic interaction with antioxidants protecting against TSE effects on growth. Thus, components of tobacco smoke perturb neurodifferentiation through mechanisms that cannot be attributed to the individual effects of nicotine, oxidative stress or interference with one-carbon metabolism. Consequently, attempted amelioration strategies may be partially effective at best, or, as seen here, can actually aggravate injury interfering with normal developmental signals and/or by sensitizing cells to TSE effects on neurodifferentiation.

In Vitro

The Satterwhite lab designed 2D live cell in vitro assays to determine exposure phenotypes in human neural stem cells (hNSCs), the commercially available progenitor cells derived from the approved WA09 embryonic stem cell line that are capable of both proliferation and differentiation to neurons, astrocytes or oligodendrocytes. The first study details nicotine and tobacco smoke extract (TSE) exposure phenotypes and phenotypic remediation by co-treatment with ascorbic acid (Horrow C, Darby MK, Satterwhite LL. Nicotine and tobacco smoke extract exposure phenotypes are remediated by ascorbic acid in human neural stem cells: a new in vitro model of neurodevelopmental toxicity, in preparation). Exposure phenotypes were scored during proliferation (48 hours), early differentiation (8 days) and mature differentiation (23 days) and an interesting interplay discovered between nicotine and the major ascorbic acid transporter SVCT2 (SLC23A2, Solute Carrier Family 23 (ascorbic acid transporter), member 2), which is required for neurodevelopment. For example, treatment of hNSCs with 10 mM nicotine reduced the number of mitotic cells in proliferating cultures 6-fold compared to controls at 48 hours (3.8% to 0.6%); no mitotic cells were found following exposure to 10 mM nicotine in TSE. Treatment of control hNSCs with 10 mM ascorbic acid (AA) decreased the number of mitotic cells 2.8-fold compared to untreated hNSCs (1.7% to 0.6%); however, AA showed no effect on mitosis in nicotine or TSE exposed cells. In contrast, apoptosis (cell death) in control hNSCs was increased 6-fold by treatment with 10 mM nicotine (0.6% to 3.6%) and increased 26-fold by treatment with 10 mM nicotine in TSE (0.6% to 16%); remarkably, co-treatment with 10 mM AA decreased apoptosis in nicotine-exposed hNSCs 5-fold (3.6% to 0.7%) and 17-fold in TSE exposed hNSCs (16% to 0.9%). These results suggest that AA may affect two independent pathways resulting in improved viability following exposure to nicotine or TSE and by either a cell cycle block and/or induced differentiation in control hNSCs. To identify the molecular mechanisms, a panel of qPCR assays was developed to score seven parameters: 1) cell cycle progression, 2) cell fate specification, 3) synaptic phenotype, 4) axonal guidance, 5) ascorbic acid-associated transcription, and genes associated with 6) tobacco use and ADHD. Early findings are that expression of the neural progenitor marker NES (Nestin) increases earlier in nicotine-exposed hNSCs compared to controls, and that nicotine or TSE significantly reduce AA induction of SVCT2; high intracellular levels of AA are critical for proper neurodevelopment.

Supplemental Project: Sex Differences in Cholinergic Bases of Memory: Hippocampal and Frontal Cortical Nicotinic and Muscarinic Mechanisms.

Data collection for muscarinic and nicotinic antagonist infusions (scopolamine and mecamylamine into the hippocampus and frontal cortex of male and female rats is done. Male rats have fewer working memory errors than female rats. Hippocampal mecamylamine (nicotinic antagonist) infusions impair males more than females. Hippocampal scopolamine (muscarinic antagonist) infusions impair females more than males. Results from the frontal cortex are mixed.

Significance: The rat studies show the cause-and-effect relationship between developmental exposure to tobacco smoke constituents and locomotor hyperactivity as well as cognitive impairment in the novel object recognition test with the tobacco smoke extract causing a more substantial effect than nicotine alone. The in vivo and neurochemical studies demonstrated the that the same tobacco smoke extract exposure disrupted dopaminergic, serotonergic and cholinergic neurotransmitter systems with greater effects of the tobacco constituent mixture compared with nicotine alone. The second half of gestation appears to be the sensitive window for persisting behavioral impairment. The in vitro studies showed cellular disruptions of proliferation and differentiation again with the tobacco constituent mixture causing more pervasive effects than the same concentration of nicotine alone. The human neural stem cells are more sensitive to both nicotine and tobacco smoke extract than the rat PC12 cells and provide an opportunity to establish direct concordance between human clinical samples (Project 1) and in vitro exposure phenotypes (Project 2).

Project 3

Aim 1: Identify ETS-related methylation targets. This is an ongoing objective. The cohorts of rats to be used for this aim have been generated (breeding, exposure, behavioral measures and tissue collection) as part of the aims of Project 2.

Our specific objectives are as follows:

  • Pool equal amounts of frontal cortex and peripheral blood genomic DNA from a total of twelve adult rats in four groups: 1) rats exposed to placebo in utero to serve as controls, 2) rats exposed in utero to nicotine levels equivalent to that of ETS, 3) rats exposed in utero to nicotine levels equivalent to that of active maternal smoking, and 4) rats exposed in utero to tobacco smoke extract with nicotine levels equivalent to that of active maternal smoking
  • Ship pooled specimens to the University of Southern California Epigenome Center for bisulfite modification and high throughput sequencing
  • At Duke, align the paired-end reads to the most current in silico bisulfite modified Rattus norvegicus reference genome (March 2012; RGSC 5.0/rn5)
  • Calculate methylation levels at individual base pair locations
  • Test for association between methylation levels and outcome variables
  • Technical validation for top hits by bisulfite pyrosequencing

This work continues to be ongoing. We submitted rat frontal cortex, hippocampus and peripheral blood tissues (as pooled samples for each exposure group with N=12 in each group) for whole genome bisulfite sequencing.

Aim 2: Identify ETS-altered methylation-expression relationships in frontal cortex. The cohorts of rats to be used for this aim have been generated (breeding, exposure, behavioral measures, tissue collection) as part of Project 2.

Our specific objectives are as follows:

  • Purify RNA from snap frozen frontal cortex; pool for transcriptome analysis
  • Assess RNA for quality and integrity
  • Submit RNA pools to the Duke IGSP Sequencing Facility for fragmentation of poly A tailed RNA, cDNA synthesis, end repair, addition of 3’ A ends, adapter ligation, PCR amplification, library validation and cluster generation
  • Align reads to the rat reference genome and to an exon-exon database
  • Combine aligned sequence reads to estimate transcript abundances
  • Validate top hits using real time RT-PCR
  • Identify those transcripts that are most strongly associated with our outcomes
  • Compare the group of differentially expressed genes to the methylation levels detected from the analysis in Aim 1

We have prepared the RNA (simultaneous with the DNA used for WGBS), assessed QA/QC metrics and pooled equal amounts of RNA from each of the twelve rats comprising a sample from frontal cortex as well as from the hippocampus of the rats. Pooled RNAs were submitted to the University of Southern California Sequencing Facility for RNA-Seq.

RNA Seq data has been generated from the same rat hippocampal and frontal cortex tissues as is being used in Aim 1 for whole genome bisulfite sequencing. The statistics Master’s students are working under the direction of Dr. Joseph Lucas to align the reads to the rat transcriptome and to quantify levels of expression.

Aim 3: Determine if DNA methylation varies with ETS dose in humans.

Our specific objectives are as follows:

  • Generate methylation data in humans for 12 loci including those identified as top candidates (ADRA2A, DRD2, MAOA, SLC6A2, MEG3 [2 regions], MMP9 and OPRD1) as well as genes identified in Project 1 and/or from our preliminary data as top candidates (IGF2/H19 [2 regions], MEG3 [2 regions], NGF and BDNF), and 20 additional loci that are identified from Aims 1 and 2
    • Umbilical cord blood from 800 NEST children, selecting those with known ETS exposure and matching to controls with no reported ETS exposure
    • Peripheral blood specimens collected postnatally from the children being followed in Project 1
  • Analyze rat frontal cortex and peripheral blood from Project 2, to validate the findings from Aim 1
  • Analyze methylation in cultured neuronal cells from Project 2, to determine if genes identified from Aim 1 and candidate genes exhibit methylation changes that affect differentiation as a result of nicotine and ETS exposure
  • Ship umbilical cord blood plasma to the UCSF Clinical Pharmacology laboratory for determinations of cotinine levels
  • Genotype genes involved in detoxification (e.g., GSTP1 I105V, CYP1A1, EPHX1 Y113H SNP variants and GSTM1, GSTT1 and GSTT2 null variants) in both the mothers and the infants
  • Obtain blood lead levels for the children being followed in Project 1 using peripheral blood specimens collected at 3-5 years of age.
  • Obtain gestational blood lead levels for the mothers of the children being followed in Project 1
  • Determine if relationships exist between the level of methylation measured and cord blood cotinine levels; results will be analyzed by gender
  • Determine relationships between genotypes of detoxification genes, DNA methylation, and exposure to environmental tobacco smoke
  • Analyze the methylation data in the children from Project 1 with respect to prenatal maternal lead levels and lead levels in the child at 3-5 years of age

We have worked with Project 1 to develop a database in which the methylation data generated for the human cohort are integrated with the epidemiologic and clinical data, including the smoke exposure variables. These data are currently undergoing multivariate analysis to determine if relationships exist between self-reported smoke exposure during pregnancy and DNA methylation at MMP9, SLC6A2, MEG3 and MEG3-IG.

The Sequenom EpiTYPER system was implemented to screen for differential methylation in four genes in rat cerebellar tissue (slc6a2, slc18a1, slc18a2 and th) involved in monoamine biosynthesis and transport. slc18a1 and slc18a2 encode for vesicular monoamine transporters 1 and 2, respectively. These transporters are involved in capturing cytosolic monoamines and packaging into vesicles for release during exocytosis or action potentials. th encodes a tyrosine hydroxylase gene. th is the rate-limiting enzyme that catalyzes the reaction converting tyrosine to l-dopa, the precursor to dopamine. Three loci (slc6a2-region2, slc18a1-region4 and slc18a2-region2) were identified for more quantitative analysis on the Pyrosequencing platform. Pyrosequencing assays for slc6a2, slc18a1 and slc18a2 have been developed. Optimization and calibration of the assay currently is in progress.

Cotinine levels have been generated for ~800 mothers of children who are participating in or are potential participants in NICHES Project 1. Cotinine levels vary widely, and there are few pregnant women who have no measurable cotinine in their blood.

Whole Genome Bisulfite Sequencing methylation data from the rat experiments in Project 2 currently are being generated; preliminary analysis indicates that even low level nicotine exposure has a substantial effect, but this needs further validation.

We have significant findings from targeted gene methylation analysis for DRD4 methylation in relation to self-report of smoke exposure by the mothers during pregnancy. Babies born to smoke-exposed mothers have about 5% lower methylation than those born to mothers reporting no exposure. We have not yet analyzed these data using the measured cotinine levels nor with respect to behavioral outcomes as determined by Project 1.

Significance: We have shown changes in DNA methylation in umbilical cord blood resulting from tobacco smoke exposure at two ADHD-relevant genes in unadjusted analysis. Adjusted analysis is underway. This supports that epigenetic alterations are associated with in utero exposure to tobacco smoke. We have determined the optimal methodology for simultaneously purification of DNA and RNA and are beginning to prepare the brain and blood from the in utero exposed/unexposed rats for whole genome and transcriptome analyses.

Future Activities:

Project 1:

We have been steadily recruiting and expect to be on target to achieve the aims. We plan to collected data for the first wave (of 2 waves) of recruitment until we reach 350 participants (we estimate this will occur in Spring 2017).  The second wave of recruitment has already started and will continue through Spring 2018. We already have started to perform preliminary analyses on Wave 1 data for publications. In addition, we have started an additional study (NICHD 1R01HD084487-01; MPIs: Fuemmeler & Kollins) examining executive functioning development and risk for obesity in this same cohort. The activities of the two projects are non-overlapping, but have allowed for more effective recruitment and retention activities.

Project 2:

Aim 1: Finish neurobehavioral assessment for all cohorts in the windows study of attention, learning, spatial working and reference memory, non-spatial memory, anxiety, fear and anxiety for male and female rats exposed throughout early 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 higher dose of nicotine (2 mg/kg/day) modeling the nicotine exposure from primary smoking. We are conducting a study of the interaction of nicotine and the prototypic polyaromatic hydrocarbon in tobacco smoke benzo-a-pyrene for gestational neurobehavioral toxicology. As previously there will be assessment of cholinergic and catecholaminergic neurotransmitter systems and a spectrum of behavioral functions including locomotor activity, anxiety, fear and cognitive function.  Determine the most sensitive neurobehavioral effects. In the third major study test rescue and therapeutic treatments. Test the efficacy of antioxidant and methyl donor rescue of TSE exposure induced neurobehavioral effects. Assess pharmacotherapies (methylphenidate, amphetamine and atomoxetine) to attenuate the neurobehavioral dysfunction caused by TSE and nicotine exposure during development.

Aim 2: Perform brain regional dissections in an age range from birth through weaning, adolescence and adulthood of male and female rats exposed in discrete developmental windows, to TSE at a dose modeling environmental tobacco smoke exposure (0.2 mg/kg/day) in comparison with vehicle control. Perform brain region specific neurochemical analyses characterizing monoaminergic and cholinergic neurotransmitter systems of in vivo tissues from TSE administration. Elucidate mechanisms underlying the TSE effects on phenotypic fate (neurons, astroglia, oligodendrocytes) in rat neural stem cells. Prepare TSE exposed samples from in vivo and in vitro studies for epigenetics evaluations.

Aim 3: The mechanistic qPCR survey will identify key genes in hNSCs that will be assayed for nicotine/TSE-induced DNA methylation changes in vitro and concordant changes explored in human clinical samples from Project 1/3 and in banked fetal tissues. A novel gender-specific hNSC in vitro model will be developed through selections from male WA01 and female WA09 hESCs. hESC exposure phenotypes also will be determined. A 3D model of hNSCs (brainoids) will be developed to test how architecture affects genomic and epigenetic response. In parallel, the new hNSC models will be evaluated using quantitative phase microscopy to identify label-free optical signatures to score living cells in real time as they differentiate.

Project 3:

Aim 1: conduct data analysis to identify targets of methylation changes due to prenatal exposure to nicotine or tobacco smoke extract and begin the process of validation in the individual rats and extend the results to the human cohort.

Aim 2: the same rat tissues from Aim 1 were used for pooled RNA transcriptome profiling to identify gene expression changes that occur in frontal cortex and hippocampus as a result of prenatal exposure to nicotine or tobacco smoke extract. Expression changes will be validated in the individual rats. These data will also be integrated with the whole genome bisulfite sequencing data from Aim 1 to identify top candidate genes (i.e., those showing a methylation change from prenatal exposure to nicotine or tobacco smoke extract and concordance in methylation change between brain and blood and that also show a correlation between methylation and expression in control versus exposed animals) for analysis in our human cohort in Aim 3.

Aim 3: we will continue to examine candidate targets of methylation in exposed humans and rats and relate this to level of exposure.  This will shift to examination of candidates identified from results obtained by Aims 1 and 2 once the data analysis is completed. We plan to generate the genotyping data as well as the lead exposure data for the mothers and children in the coming year.

We will continue to bank and track biological specimens as they are collected from Projects 1 and 2.

References:

Hall BJ, Cauley M, Burke D, Kiany A, Slotkin TA, Levin ED. Cognitive and behavioral impairments evoked by low level exposure to tobacco smoke constituents: comparison with nicotine alone. Toxicologial Sciences 2016;15:236-244.


Journal Articles: 33 Displayed | Download in RIS Format

Other center views: All 116 publications 38 publications in selected types All 33 journal articles
Type Citation Sub Project Document Sources
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)
  • Full-text from PubMed
  • Abstract from PubMed
  • Associated PubMed link
  • Full-text: EHP-Full Text HTML
  • Abstract: EHP-Abstract
  • Other: EHP-Full Text PDF
  • 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)
  • Abstract from PubMed
  • Full-text: ScienceDirect-Full Text HTML
    Exit
  • Abstract: ScienceDirect-Abstract
    Exit
  • Other: ScienceDirect-Full Text PDF
    Exit
  • 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)
  • Abstract from PubMed
  • Full-text: Mary Ann Liebert-Full Text HTML
    Exit
  • Abstract: Mary Ann Liebert-Abstract
    Exit
  • Other: Mary Ann Liebert-Full Text PDF
    Exit
  • 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)
  • Full-text from PubMed
  • Abstract from PubMed
  • Associated PubMed link
  • Full-text: VCU-Abstract and Full Text PDF
    Exit
  • Abstract: Sage Journals-Abstract
    Exit
  • 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)
  • Full-text from PubMed
  • Abstract from PubMed
  • Associated PubMed link
  • Full-text: BMC-Full Text HTML
    Exit
  • Abstract: BMC-Abstract
    Exit
  • Other: BMC-Full Text PDF
    Exit
  • 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)
  • Abstract from PubMed
  • Abstract: Oxford Journals-Abstract
    Exit
  • 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)
  • Full-text from PubMed
  • Abstract from PubMed
  • Associated PubMed link
  • Abstract: ScienceDirect-Abstract
    Exit
  • Other: ResearchGate-Abstract
    Exit
  • 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)
  • Abstract from PubMed
  • Abstract: Taylor & Francis-Abstract
    Exit
  • 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)
  • Abstract from PubMed
  • Full-text: ScienceDirect-Full Text HTML
    Exit
  • Abstract: ScienceDirect-Abstract
    Exit
  • Other: ScienceDirect-Full Text PDF
    Exit
  • 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)
  • Full-text from PubMed
  • Abstract from PubMed
  • Associated PubMed link
  • Full-text: MDPI-Full Text HTML
    Exit
  • Abstract: MDPI-Abstract
    Exit
  • Other: MDPI-Full Text PDF
    Exit
  • 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)
    R835437 (2014)
  • Full-text from PubMed
  • Abstract from PubMed
  • Associated PubMed link
  • Full-text: Karger-Full Text HTML
    Exit
  • Abstract: Karger-Abstract
    Exit
  • Other: Karger-Full Text PDF
    Exit
  • 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)
  • Full-text from PubMed
  • Abstract from PubMed
  • Associated PubMed link
  • Full-text: ScienceDirect-Full Text HTML
    Exit
  • Abstract: ScienceDirect-Abstract
    Exit
  • Other: ScienceDirect-Full Text PDF
    Exit
  • 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)
  • Full-text: Oxford Journals-Full Text PDF
    Exit
  • Abstract: Oxford Journals-Abstract & Full Text HTML
    Exit
  • Journal Article Schechter JC, Kollins SH. Prenatal smoke exposure and ADHD: advancing the field. Pediatrics 2017;139(2):e20163481 (2 pp.). R835437 (2017)
  • Full-text from PubMed
  • Abstract from PubMed
  • Associated PubMed link
  • Full-text: Pediatrics-Full Text HTML
    Exit
  • Abstract: Pediatrics-Abstract
    Exit
  • Other: Pediatrics-Full Text PDF
    Exit
  • 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)
  • Full-text from PubMed
  • Abstract from PubMed
  • Associated PubMed link
  • Full-text: MDPI-Full Text HTML
    Exit
  • Abstract: MDPI-Abstract
    Exit
  • Other: MDPI-Full Text PDF
    Exit
  • 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)
    R835437 (2014)
  • Full-text from PubMed
  • Abstract from PubMed
  • Associated PubMed link
  • Full-text: EHP-Full Text PDF
  • Abstract: EHP-Abstract & Full Text HTML
  • 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)
  • Full-text from PubMed
  • Abstract from PubMed
  • Associated PubMed link
  • Full-text: ScienceDirect-Full Text HTML
    Exit
  • Abstract: ScienceDirect-Abstract
    Exit
  • Other: ScienceDirect-Full Text PDF
    Exit
  • 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)
    R835437 (2014)
  • Full-text from PubMed
  • Abstract from PubMed
  • Associated PubMed link
  • Full-text: ScienceDirect-Full Text HTML
    Exit
  • Abstract: ScienceDirect-Abstract
    Exit
  • Other: ScienceDirect-Full Text PDF
    Exit
  • 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)
  • Abstract from PubMed
  • Full-text: ScienceDirect-Full Text HTML
    Exit
  • Abstract: ScienceDirect-Abstract
    Exit
  • Other: ScienceDirect-Full Text PDF
    Exit
  • 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)
  • Abstract from PubMed
  • Abstract: Oxford Journals-Abstract
    Exit
  • 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)
  • Abstract from PubMed
  • Full-text: ScienceDirect-Full Text HTML
    Exit
  • Abstract: ScienceDirect-Abstract
    Exit
  • Other: ScienceDirect-Full Text PDF
    Exit
  • 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)
  • Full-text from PubMed
  • Abstract from PubMed
  • Associated PubMed link
  • Full-text: ScienceDirect-Full Text HTML
    Exit
  • Abstract: ScienceDirect-Abstract
    Exit
  • Other: ScienceDirect-Full Text PDF
    Exit
  • 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)
  • Full-text from PubMed
  • Abstract from PubMed
  • Associated PubMed link
  • Full-text: Oxford Press-Full Text HTML
    Exit
  • Abstract: Oxford Press-Abstract
    Exit
  • Other: Oxford Press-Full Text PDF
    Exit
  • 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)
    not available
    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)
  • Full-text from PubMed
  • Abstract from PubMed
  • Associated PubMed link
  • Full-text: MDPI-Full Text HTML
    Exit
  • Abstract: MDPI-Abstract
    Exit
  • Other: MDPI-Full Text PDF
    Exit
  • 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)
  • Full-text from PubMed
  • Abstract from PubMed
  • Associated PubMed link
  • Full-text: Wiley-Full Text HTML
    Exit
  • Abstract: Wiley-Abstract
    Exit
  • Other: Wiley-Full Text PDF
    Exit
  • 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)
  • Abstract from PubMed
  • Abstract: Epigenomics-Abstract
    Exit
  • Other: ResearchGate-Abstract
    Exit
  • 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)
  • Full-text from PubMed
  • Abstract from PubMed
  • Associated PubMed link
  • Full-text: Genetics & Epigenetics-Full Text PDF
    Exit
  • Abstract: Genetics & Epigenetics-Abstract
    Exit
  • Other: ResearchGate-Full Text PDF
    Exit
  • 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)
    not available
    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)
    not available
    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)
    not available
    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)
    not available
    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)
    not available

    Supplemental Keywords:

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

    Relevant Websites:

    NICHES web Site: NiCHES: A Children‘s Environmental Health and Disease Prevention Research Center, Duke University Exit

    Science Education web site: RISE at Duke | Raising Interest in Science Education: Research & Development by Dr. Rochelle Schwartz-Bloom Exit

    COTC web site: Help Babies Avoid Smoke! Exit

    Progress and Final Reports:

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