Prenatal Bisphenol A (BPA) exposure on epigenetic changes in

fetal mouse tissues

Alice DouglasBartolomei Lab

STEER Summer 2016

The DOHaD Hypothesis: • Developmental Origins of Health and Disease-

– focuses on fetal origins of adult disorders that are influenced by the interaction of genetic background and environmental exposures such as nutrition, stress, pharmaceuticals and toxins

TIME Magazine 2010 Xin et al 2015

F1

Environmental Exposures and Health

Disease

Drugs

Diet

ExerciseStress

Chemicals

Bisphenol A (BPA)

www.ansci.wisc.edu

Where is it found?

www.wordpress.vermontlaw.edu

BPA as an endocrine disruptor

• Estrogen mimic

• Proposed modes of action– Genomic– Non-genomic

• Inappropriately activates or inhibits a cellular response

NIEHS

(Estrogen)

(BPA)

(ER)

What is the Mechanism?

Epigenetic Changes

DrugsDiet

ChemicalsExercise

Stress

Disease

Gene Expression

Epigenetics

• epi-from the Greek, “upon,” “on,” or “above” • chemical and structural modifications on DNA

that may impact gene expression– Example: DNA methylation

http://www.delawareneuroscience.org/Pages/Roth.htm

Previous Work in the Bartolomei Lab:

Metabolic changes in adulthood:• F1 males: fatter, glucose intolerant• F1 females: not affected

Behavior changes in adulthood:• F1 males: depressed-like state • F1 females: not tested

Susiarjo et al (2015) EndocrinologyUnpublished

Objective:

• Physiological changes in adult animals prompted search for molecular changes in relevant tissues during early development

BPA Exposure

DNA Methylation Changesin Liver and Brain?

Metabolic and Behavior Phenotype

Prenatal Exposure Paradigm

Xin et al 2015

www.ansci.wisc.edu

www.nzrr.org

Lower BPA- 50 μg/kg in feed (estimated 10 μg per kg body weight per day) Upper BPA-50 mg/kg in feed (estimated 10 mg per kg body weight per day)

Strengths of Experimental Set Up:

• Dosage• Route of exposure• Window of exposure

Dissection (F1, E12.5-13.5 and E17.5-18.5) Fetal liver, brain and placenta

RNA extraction

RFLP-allele specific PCR

DNA extraction

LUMA

SMC PCR (n=183)

LUMA

• Luminometric Assay (LUMA)-global DNA methylation– Restriction Enzymes: • MSPI: methylation insensitive • HPAII: methylation sensitive• Ratio of HPAII to MSPI gives % Methylation

www.epigenome.dept.showa.gunma-u.ac.jp

Comparing Liver and Brain: Mid-gestation Female

Control n=6(3) Lower Dose n=10(4) Upper Dose n=12(5)Average= 57.16% Average= 59.92% Average= 49.85%

Control n=6(4) Lower Dose n=6(2) Upper Dose n=7(4) Average= 68.49% Average= 69.62% Average= 68.37%

% M Liver

% M Brain

Each diamond=one embryoEach color=one litter

n.s.n.s.

n.s.n.s.

Statistical Test: t-test

Comparing Liver and Brain: Mid-gestation Male

Control n=7(4) Lower Dose n=12(4) Upper Dose n=17(6)Average= 50.96% Average= 60.01% Average= 59.08%

Control n=4(2) Lower Dose n=8(3) Upper Dose n=5(4) Average= 72.67% Average= 70.14% Average= 72.45%

% M Liver

% M Brain

n.s.n.s.

n.s.n.s.

Comparing Liver and Brain: Term Female

% M Brain% M Liver

Control n=11(3) Lower Dose n=13(3) Upper Dose n=3(2) Average= 59.04% Average= 53.12% Average= 54.92%

Control n=5(2) Lower Dose n=7(3) Upper Dose n=3(2) Average= 70.407% Average= 68.04% Average= 72.50%

n.s.

p < 0.05

n.s.n.s.

Comparing Liver and Brain: Term Male% M Liver % M Brain

Control n=6(3) Lower Dose n=12(4 Upper Dose n=8(2) Average= 61.31% Average= 57.39% Average= 51.75%

Control n=4(2) Lower Dose n=8(3) Upper Dose n=3(1) Average= 70.77 % Average= 67.70% Average= 71.27%

p < 0.05n.s.

n.s.n.s.

Conclusions:

• Tissue, sex, dose and age specific changes in response to BPA exposure– Female term liver DNA methylation is significantly

reduced in the lower dose exposure group– Male term liver DNA methylation is significantly

reduced in upper dose exposure group

BPA Exposure

Metabolic and Behavior Phenotype

DNA Methylation Changes

Is this causal?

Future Directions of Research:

• Adult tissue • Other compounds– combining compounds for more realistic multi-compound exposures

• Site specific DNA methylation and total expression changes – Dnmt expression level changes– Expression of Liver and Brain function genes

Acknowledgements:• The STEER Program: – Dr. Jeff Field– Dr. Maria Antonia-Andrews– Dr. Rich Pepino– Dr. Marilyn Howarth

• Dr. Marisa Bartolomei– Frances Xin– Erin Fischer– Jen Myers– Martha Stefaniak– The rest of the Bartolomei lab!

Works Cited:• DeBenedictis B, Guan H, Yang K. Prenatal Exposure to Bisphenol A Disrupts Mouse Fetal Liver

Maturation in a Sex-Specific Manner. Journal of Cellular Biochemistry 2015; 117:344-50.• Calafat AM, Kuklenyik Z, Reidy JA, Caudill SP, Ekong J, Needham LL. Urinary concentrations of

bisphenol A and 4-nonylphenol in a human reference population. Environmetnal Health Perspectives 2005;113:391–5.

• Gioiosa L, Palanza P, Parmigiani S, Vom Saal FS. Risk Evaluation of Endocrine-Disrupting Chemicals: Effects of Developmental Exposure to Low Doses of Bisphenol A on Behavior and Physiology in Mice (Mus musculus). Dose-Response 2015; 13:1-8.

• Hijazi A, Guan H, Cernea M, Yang K. Prenatal exposure to bisphenol A disrupts mouse fetal lung development. Faseb Journal 2015; 29:4968-77.

• Susiarjo M, Xin F, Bansal A, Stefaniak M, Li C, Simmons RA, Bartolomei MS. Bisphenol A Exposure Disrupts Metabolic Health Across Multiple Generations in the Mouse. Endocrinology 2015; 156:2049-58.

• Susiarjo M, Sasson I, Mesaros C, Bartolomei MS. Bisphenol A Exposure Disrupts Genomic Imprinting in the Mouse. PLOS Genetics 2013; 9:1-18.

• Whitehead R, Guan H, Arany E, Cernea M, Yang K. Prenatal exposure to bisphenol A alters mouse fetal pancreatic morphology and islet composition. Hormone Molecular Biology and Clinical Investigation 2016; 25:171-9.

• Xin F, Susiarjo M, Bartolomei MS. Multigenerational and transgenerational effects of endocrine disrupting chemicals: A role for altered epigenetic regulation? Seminars in Cell & Developmental Biology 2015; 43:66-75.

Questions?

Thanks!