Impact and Outcomes of MoBa Contaminant Studies

Stephanie M. Engel, PhD

Associate Professor of Epidemiology

Impact and Outcomes of MOBA Environmental Studies: Outline

Impact of MoBa environmental studies

Highlight major MoBa contributions to environmental research

Preliminary data from ongoing MoBa environmental research on phthalates and Attention-deficit Hyperactivity Disorder (ADHD)

Environmental Research has been a major focus of studies published to date

Environmental Exposures Examined in MoBa to date

Smoking (Active & Passive)

Perfluoroalkyl substances

Air pollution

PAHs (dietary)

PCBs, dioxin (measured and dietary)

meHg (dietary)

Acrylamide (dietary)

BPA

Phthalates

Triclosan

Flame retardantsCell phone use (non-

ionizing Radiation)

What are the attributes of MoBa that favor environmental research?

Availability of biospecimens in the prenatal window for the measurement of biomarkers of exposure or biological mechanism

Ability to link with disease registries to study rare outcomes

Questionnaire & genetic data can be used to investigate modifiers of exposure-outcome associations (or as predictors or estimators of exposure themselves)

What MoBa Environmental Research has had the biggest impact?

Hard to judge impact in the short term

Number of citations (increase over time)

Replication in independent studies (increase over time)

Using Web of Science…

1. DNA Methylation as a biomarker of maternal smoking

2. PFAS in relation to fertility and pregnancy complications

Epigenome is non-sequence based modifications of DNA and histone proteins that

are heritable

Impact on the functioning of our genome

The prenatal period may be uniquely susceptible to epigenomic modifications

CpGs in AHRR and CYP1A1differentially methylated in relation to maternal smoking (p < 10-7)

Smoking validated by urinary cotinine

Huge impact- 3rd most cited MoBa article, 231 citations since 2012, top 1% of papers published that year

Proof of principle that epigenomemay be a catalogue for environmentally induced disease

13 cohorts including over 6000 people (Pregnancy and Childhood Epigenetics Consortium- PACE)

Over 6,000 CPGs differentially methylated

Top methylation mark AHRR p = 1.64x10-193

Associations persisted in cohorts with later childhood DNA, suggesting stability of biomarker over time

Perfluorinated Compounds

Man made chemicals used in commercial and industrial applications

Relatively long half-lives, although this varies to some extent across substances

Widespread distribution in the environment

Diet is likely a major exposure route

Evidence of reproductive toxicity and other health effects in animal studies

MoBa has been a major contributor to the PFAS literature on human health effects At least 11 published papers addressing measurement,

pharmacokinetics and health effects

Spanning multiple disease areas Birthweight and preterm birth Preeclampsia Subfecundity and time-to-pregnancy (subfecundity

paper in top 1% of citations for that year) Immunotoxicity Thyroid hormone Clinical chemistries

Cross-sectional of 891 pregnancies between 2003–2004

Non-fasting mid-pregnancy plasma samples analyzed for nineteen PFASs, total cholesterol, high-density lipoprotein (HDL) cholesterol, low-density lipoprotein cholesterol, and triglycerides

Five of the seven PFASs studied were positively associated with HDL cholesterol, and all seven had elevated HDL associated with the highest quartile of exposure.

Serum PFOS has also been positively associated with HDL cholesterol among: Non-pregnant Inuit adults (Chateau-Degat et al., 2010)

Children and adolescents from a highly PFOA-exposed community in Ohio and West Virginia (Frisbee et al., 2010).

Use of MoBa Dietary Information to EstimateExposure to Environmental Contaminants

Diet is a major route of exposure to many contaminants, including PCBs, dioxin, meHG

Food frequency questionnaire completed at mid-pregnancy for the entire MoBa cohort

Allows entire cohort to be included, rather than nested sample required for biomarker studies

Can validate estimates on a subset with biospecimen-based biomarker measures

Example of leveraging entire MoBa based on estimates of exposure (n = 46, 750)

Increased dietary meHG, increased odds of unintelligible speech, and weaker communication skills

Patterns of food intake may vary across geographic locations which could impact diet quality and exposure to toxicants, so generalizability is always a concern.

New Approaches for Critical Windows-MoBa tooth bank

FIGURE 2 . Lead bio-image in a macaque first permanent molar. (a) Clear transitions in lead levels (as 208 Pb: 43 Ca) can be seen in the elemental bio-image. Color index represents the range in lead levels from low (dark blue) to high (red). (b) Developmental time from birth (horizontal axis) and lead levels in discrete sampling points in dentine along the enamel-dentine junction (vertical axis). Lead levels are low in prenatal dentine and then increase sharply after birth to reach a maximum at approximately day 106. Crown of the mesiobuccal cusp was completed at age 267 days for this animal. High levels of lead are also visible in secondary dentine near the pulp chamber.

Manish A et al. Current Opinion in Pediatrics. 25(2):261-267, April 2013

Prenatal Toxicant Exposures and Attention-Deficit Hyperactivity Disorder

Funded in part by NIEHS R01 ES021777

Study TeamUniversity of North Carolina at

Chapel HillStephanie Engel

Duke UniversityAmy Herring

National Institute of Environmental Health Sciences

Matthew LongneckerJane Hoppin (NCSU)

University of Massachusetts at Amherst

R. Thomas Zoeller

Norwegian Institute of Public HealthHeidi Aase

Ted Reichborn-KjennerudGro Villanger

Gun Peggy Knudsen

Oslo University HospitalPal Zeiner

Additional CollaborationsCathrine Thomsen, NIPH

Amrit Sakhi, NIPHEnrique Cequier, NIPH

Specific Aims

1. To examine association of prenatal phthalate exposure with preschool and childhood ADHD◦ Modification by child sex

◦ Mediation by maternal or child thyroid hormone disruption

2. To examine association of prenatal organophosphate pesticide exposure and preschool and childhood ADHD◦ Modification by maternal or child metabolism enzyme

genetic polymorphisms

What are Phthalates? High-production volume chemicals: over three

million metric tons produced annually

High molecular weight phthalates are used to make plastics flexible, transparent and durable, and they are also used in food packaging, building materials and toys

Low molecular weight phthalates are often found in personal care products, solvents, adhesives and enteric coatings on medications

Exposure can occur through inhalation, dermal absorption, ingestion, and intravenous exposure

Phthalate ExposureInternationally, population exposures are ubiquitous and high

Used in a wide range of consumer products: plasticizers, fragrance carriers, solvents, adhesives and lubricants

Exposure can occur through the diet (DEHP), as well as through inhalation, dermal absorption, and through injection.

Why Study Phthalates in relation to ADHD?

Animal studies have linked gestational phthalate exposure with:

• Anxiety

• Hyperactivity

• Impacts on learning & memory

Prospective studies of prenatal phthalate exposure and child development have found associations with:

• Inattention

• Hyperactivity

• Conduct problems

• Emotional reactivity

• Impaired working memory

No study has had a clinically assessed outcome

We measured maternal prenatal urinary phthalate concentrations in mothers at 17 weeks’ gestation

Urinary concentrations were measured at the NIPH in CathrineThomsen’s lab

Urinary dilution accounted for by specific gravity

Almost all metabolites were 100% detectable (oxo-MiNP98.5%)

Phthalates are moderately correlated (because they are in everything)

Prenatal Phthalates, Maternal Thyroid Hormones, and Risk of Attention-Deficit Hyperactivity Disorder in the Norwegian Mother and Child Cohort

Stephanie M. Engel, PhD1; Gro D. Villanger, PhD2; Rachel C. Nethery1; CathrineThomsen2; Amrit K. Sakhi2 ; Samantha S.M. Drover, MSc1; Jane A. Hoppin, ScD3; Pal Zeiner, MD4; Gun Peggy Knudsen, PhD2; Ted Reichborn-Kjennerud, MD2, PhD5; Amy H. Herring, ScD6; Heidi Aase, PhD2

Phthalates & Childhood ADHD In Norway (NPR diagnosis)

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Bayesian multipollutant model including all phthalates, and additionally adjusted for child sex, mother’s age, mother’s education level, mother’s marital status, mother’s smoking status, parity, maternal depression during pregnancy, and year, and standardized to specific gravity

Work in progress by Elizabeth Kamai, PhD candidate UNC Chapel Hill

Prenatal Phthalates and Preschool ADHD in the Norwegian Mother and Child Cohort

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Phthalates & Preschool ADHD

Models adjusted for specific gravity, batch, child sex, maternal age (continuous), education, marital status, smoking during pregnancy, parity, and co-adjusted for all other phthalates 27

Reconciling preschool and childhood ADHD findings Preschool ADHD may under-represent children

with inattentive symptoms because that is harder to pick-up in the preschool period

Preschool ADHD diagnoses may not always persist into childhood, and therefore could have a different constellation of risk factors

Childhood ADHD, since it is registered by the NPR, may be more severe because it was brought to clinical attention by the family

Do phthalates affect neurodevelopment through maternal thyroid hormone disruption?

Phthalates (including DEHP) have been shown down-regulates transcriptional activity of NIS (human sodium/iodide symporter)

Changes in serum concentrations of triiodothyronine (T3) and thyroxine (T4) with increasing exposure to some phthalates

Thyroid hormones are essential for fetal neurodevelopment, and mothers provide all that is necessary to meet fetal demands in the first ½ of pregnancy

Both maternal hypo- and hyperthyroidism have been associated with ADHD

Is the effect of phthalates mediated by thyroid hormones?

Three arrows assessed

Phthalates and ADHD

Thyroid hormones and ADHD

Phthalates and thyroid hormones

Phthalates

Thyroid Hormones

ADHD

Maternal thyroid hormone biomarkers in MoBa

Maternal blood collected at 17 weeks’ gestation

Analyzed at ARUP Laboratories for:

Thyroid stimulating hormone (TSH)

Thyroxine (T4)

Triiodothyronine (T3)

Thyroid peroxidase antibodies

T3-uptake

Calculated Free T4 index & Free T3 index

Gestational Thyroid Hormone Concentrations and Risk of Attention-Deficit Hyperactivity Disorder in the Norwegian Mother and Child Cohort Study

*Stephanie M. Engel, PhD1; Gro D. Villanger, PhD2; Amy Herring, ScD3; Rachel C. Nethery4; Samantha Drover, MSc1; R. Thomas Zoeller, PhD5; Helle M. Meltzer, PhD2; Pal Zeiner, MD6; Gun Peggy Knudsen, PhD2; Ted Reichborn-Kjennerud, MD, PhD2,7; Matthew P. Longnecker, MD, ScD8; Heidi Aase, PhD2

Maternal Prenatal Thyroid Hormone Concentrations and Childhood ADHD

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TSH T3 FT3 T4 FT4 TPO-Ab

FT3i/FT4iRatio

Neonatal TSH and Risk of Attention-Deficit Hyperactivity Disorder in the Norwegian Mother and Child Cohort Study

*Gro D. Villanger, PhD2 and the ADHD study team

Neonatal TSH and ADHD|| || || | | | |||| ||||| | || || ||| || | || |||| |||| ||| || ||| || | ||| | || ||||| | | | ||| ||||| | || | || | | | |||| || ||| || ||| | | ||||| ||| | || || |||| | |||| || || || | | || | | || || ||| || | ||| ||| ||| | || ||| || || || |||| || ||| | | | || | | || | || | || | ||| || | ||||| | || | || |||| ||| || || || || || ||||| | ||| || || || || | || || | | || ||| | |||| || | || | | | ||| | | || || || || || | || | ||| |||| || || || | || | | || || || | ||| ||| || || |||| ||| ||| | || | ||| || || || || | || | ||| | || | | | || | | ||| | || || || || |||||| | | || || | |||| || |||| || | ||| ||| |

ADHD DiagnosisP-overall=0.048

P-interaction=0.041

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Work in progress Phthalates and Thyroid Hormones

Gro Villanger, Samantha Drover and the ADHD Study Team

Several phthalates associated with changes in circulating thyroid hormone concentrations, but not DEHP

Causal Mediation? Not that we can detect.

No Interaction Interaction

NDE NIE NDE NIE

OR 95% CI OR 95% CI OR 95% CI OR 95% CI

Thyroid Stimulating

Hormone (TSH)1

1.45 (1.11,1.88) 1.00 (0.99,1.01) 1.45 (1.12,1.88) 1.00 (0.99,1.02)

Triiodothyronine (T3)1

1.46 (1.12,1.90) 1.00 (0.98,1.02) 1.53 (1.15,2.03) 1.00 (0.96,1.05)

Thyroxine (T4)1

1.46 (1.13,1.90) 1.00 (0.99,1.01) 1.49 (1.14,1.95) 0.99 (0.97,1.02)

Preterm Delivery2

1.45 (1.12,1.88) 1.00 (1.00,1.00) 1.44 (1.11,1.87) 1.00 (0.99,1.02)

Other potential pathways Oxidative stress

Ferguson et al. found strong assocations between DEHP and oxidative stress biomarkers

Oxidative stress biomarkers also strongly associated with later preterm delivery

Preterm delivery a strong risk factor for ADHD

Mediation by oxidative stress and/or preterm delivery?

Next steps Recently received the organophosphate pesticide metabolite data,

and we have begun analyses of these data. Looking for PhD students interested in taking on some of this for a dissertation.

Samantha Drover, PhD student at UNC Chapel Hill will be analyzing the maternal and neonatal thyroid hormone concentrations in relation to Preschool ADHD and dimensional data for her dissertation

Elizabeth Kamai, PhD candidate at UNC Chapel Hill finishing a paper on prenatal phthalates and preschool ADHD.

Renewal application to NIH in development to expand the chemicals analyzed and incorporate additional case groups

AcknowledgementsADHD Study team, especially Gro Villanger, Heidi Aase, Amy Herring and Rachel Nethery

This research was funded in part by NIEHS R01ES021777 and the Intramural Research Program of the NIH, National Institute of Environmental Health Sciences. The Norwegian Mother and Child Cohort Study is supported by the Norwegian Ministry of Health and Care Services and the Ministry of Education and Research, NIH/NIEHS (contract no N01-ES-75558), NIH/NINDS (grant no.1 UO1 NS 047537-01 and grant no.2 UO1 NS 047537-06A1). The Preschool ADHD study, a substudy to MoBa, is supported by funds and grants from the Norwegian Ministry of Health, The Norwegian Health Directorate, The South Eastern Health Region, G&PJ Sorensen Fund for Scientific Research, and from The Norwegian Resource Centre for ADHD, Tourette’s syndrome and Narcolepsy. We are grateful to all the participating families in Norway who take part in this on-going cohort study.