Grantee Research Project Results

2013 Progress Report: Neurodevelopment and Improving Children's Health following EtS exposure (NICHES)

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.

 

 

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.

 

 

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:

Supplemental Keywords:

Relevant Websites:

http://niches.duke.edu Exit

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

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

Progress and Final Reports: