Early life metal exposure and epigene4cs: a transdisciplinary approach

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1 Early life metal exposure and epigene4cs: a transdisciplinary approach Alison P. Sanders, PhD Postdoctoral Fellow Department of Preven4ve Medicine Icahn School of Medicine at Mount Sinai

2 Trans- disciplinary areas: Environmental metals and epigene4cs Exposure Science Epidemiology Toxicology 2

no known cause (Brent and Beckman 1990). Preterm birth Approximately 12% of all live births (WHO 2013). $26.

3 Leading causes of infant mortality: Birth defects Approximately 3% of all live births (Petrini et al., 2002). $2.6 billion associated hospital costs in the US (Russo and Elixhauser 2006) Roughly 60-70% of birth defects have no known cause (Brent and Beckman 1990). Preterm birth Approximately 12% of all live births (WHO 2013). $26.2 billion associated medical expenditures & produc4vity losses in the US (IOM 2006) Rates of PTB are rising globally.

4 Metals are understudied environmental teratogens in human popula4ons Environmental 10% Metals rank among the top 10 in the priority list of hazardous substances Gene4c 20% Unknown 70% (ATSDR, 2013) 4

5 Metals are understudied environmental teratogens in human popula4ons Environmental 10% Metals rank among the top 10 in the priority list of hazardous substances Gene4c 20% More than 100 million people exposed worldwide to elevated levels of arsenic drinking water Unknown 70% (ATSDR, 2013) 5

6 Arsenic levels elevated in North Carolina wells Sanders et al., 2012 >7,700 had detectable arsenic (12%) Over 1,500 wells exceeded 10 μg/l (the EPA MCL) >200 exceeded 50 μg/l; maximum was 806 μg/l 6

7 Metals are understudied environmental teratogens in human popula4ons Environmental 10% Gene4c 20% Unknown 70% Evidence of metal exposure in NC pregnant women Metal % detected n Arsenic Cadmium Mercury Lead Sanders et al.,

Evidence of metal- associated defects Metals

Arsenic- treated (Domingo 1994; Golub 1998;

8 Evidence of metal- associated defects Metals cause gross morphological defects Control Arsenic- treated (Domingo 1994; Golub 1998; Holson 2000) Arsenic- and cadmium- induced heart defects (Li Ahir, Sanders et al., 2013 et al., 2012; Li et al., 2009; Cheng et al., 2001) Li et al., 2009 Epidemiological links with metals in drinking water (Zierler et al., 1988; Aschengrau et al., 1993; Engel and Smith, 1994 Brender et al., 2006; Kwok et al., 2006) Sanders et al in submission 8

9 Trans- disciplinary areas: Environmental metals and epigene4cs Exposure Science Epidemiology Toxicology 9

10 What are the biological effects of metal exposure on the heart?? Mechanism of biological pathway perturba9on 10

11 What are the biological effects of Study goal: To determine epigene4c modifiers that may modulate gene expression in metal- exposed heart cells metal exposure on the heart?? Mechanism of biological pathway perturba9on 11

12 Epigene4c mechanisms may mediate metal- induced defects Epigene4cs: The study of heritable changes that occur above the genome. Epigene9c mechanisms:» micrornas» DNA methyla4on» Histone modifica4on 12

13 An overview of cell signaling DNA DNA contains our genes mrna Protein Transcrip4on Transla4on Genes are expressed as messenger RNA Proteins carry out cell func4ons/signaling 13

are mrna expressed copies = as messenger increased RNA

14 An overview of cell signaling DNA DNA contains our genes mrna Protein Transcrip4on Transla4on Genes More are mrna expressed copies = as messenger increased RNA gene expression Proteins carry out cell func4ons/signaling 14

15 mirna are regulators of gene expression and can change cell func4on DNA DNA contains our genes mrna Protein Transcrip4on Transla4on mirna micrornas (mirnas) can silence gene expression Func9onal changes in the cell 15

mirna are regulators of gene expression and can change cell func4on DNA DNA contains our genes mrna Protein Transcrip4on mirnas 20-24 nucleo4de, single- stranded RNA Post- transcrip4onal mirna

16 mirna are regulators of gene expression and can change cell func4on DNA DNA contains our genes mrna Protein Transcrip4on mirnas nucleo4de, single- stranded RNA Post- transcrip4onal mirna regula4on micrornas that typically (mirnas) results in gene silencing can silence gene Selec4vely bind and target hundreds expression of mrna (~60% of genes) Transla4on Func9onal changes in the cell 16

17 Why are mirnas important? Exposure Changes in gene expression Altered cell signaling and func9on Altered Development (Ahir et al., 2013) 17

18 Why are mirnas important? Exposure Changes in gene expression mirna regula4on of gene expression controls aspects of cell development, prolifera4on, apoptosis, and Altered differen4a4on cell signaling and func9on Altered Development (Ahir et al., 2013) 18

19 mirnas are involved in heart development mir- 1 and mir- 133 are involved in cell prolifera4on and maintenance processes that dictate gene expression in heart development and func4on (Cordes and Srivasta, 2009; Liu and Olson, 2010). mirna are vital for normal embryonic development Control mirna dele4on Mice deficient in mir- 1 and mir 17~92 developed fatal ventricular septal defects (Zhao et al., 2007; Ventura et al., 2008). (Huang et al., 2010) 19

20 Cadmium and heart development Cd- altered heart development supported by animal studies (Cheng et al. 2001; Ronco et al. 2011) Strong evidence of associa4on between Cd and cardiovascular diseases (Tellez- Plaza et al. 2010, 2012, 2013) The mechanism of Cd- induced toxicity on the heart is not fully understood. Suggested mechanism: Indirectly genotoxic, cell cycle progression or apoptosis, transcrip4onal modifica4on? Biological pathway perturba9on 20

21 In vitro design icell Cardiomyocytes: Human- derived induced pluripotent stem cell (ips) cardiomyocytes (Cellular Dynamics Interna4onal, Madison WI) Cardiomyocytes were cultured to a state of synchronous bea4ng (144 hrs) Cells were exposed to 0.05, 0.1, or 0.5 µm CdCl 2 (5.6, 11.3, 56.3 ppb) for 24 hours then mirna and mrna were extracted. Microarrays were used to assess genome- wide mirna and mrna expression changes. 21

22 In vitro design icell Cardiomyocytes: Human- derived induced pluripotent stem cell (ips) cardiomyocytes (Cellular Dynamics Interna4onal, Madison WI) Hypothesis: Cardiomyocytes mirna- modified were cultured signaling to a state pathways of synchronous in human heart bea4ng cells are (144 altered hrs) by metal exposure Cells were exposed to 0.05, 0.1, or 0.5 µm CdCl 2 (5.6, 11.3, 56.3 ppb) for 24 hours then mirna and mrna were extracted. Microarrays were used to assess genome- wide mirna and mrna expression changes. 22

23 Measured viability and contrac4lity None of the CdCl 2 doses were cytotoxic Func4onal changes in contrac4lity Untreated control 10μM CdCl 2 23

Transla4on mrna Affymetrix GeneChip Human

24 Microarray analysis of mrna and mirna expression levels DNA mirna Agilent Human mirna Microarray V16 mrna Transcrip4on mirna 1,348 human mirnas 53,880 probesets Protein Transla4on mrna Affymetrix GeneChip Human Gene 2.0ST Array 25,968 human mrnas 24 53,617 probesets

25 Cd exposure altered expression of 8 mirnas (p<1x10-23 ) Thresholds for differen9al expression: FC - 1.5, FC 1.5 p<0.01 enzyme microrna complex / group cytokine kinase transcriptional regulator transporter other direct interaction indirect interaction 25

26 Cd exposure altered expression of 31 genes Control samples Cadmium-exposed samples 26 genes 5 genes down- regulated up- regulated FC 1.5 p<

27 Cd exposure altered expression of 31 genes Control samples Cadmium-exposed samples 26 genes 5 genes down- regulated up- regulated Family of 10 metallothioneins FC 1.5 p<

development organismal development P- value 1 10-16 The 8 mirna- associated

28 Eight differen4ally expressed genes were predicted mirna targets mirna- associated (8 genes) Top network func9ons gene expression embryonic development organismal development P- value The 8 mirna- associated genes had increased expression in the cadmium- exposed samples compared to controls 28

Poten4al mechanism of mirna- mediated Cd- toxicity in the heart mirna- associated mirna- independent Interac4ng proteins (p<1 10-26 ) G-protein coupled

29 Poten4al mechanism of mirna- mediated Cd- toxicity in the heart mirna- associated mirna- independent Interac4ng proteins (p< ) G-protein coupled receptor cytokine enzyme ion channel kinase phophatase transcriptional regulator transporter complex / group other direct interaction indirect interaction 29

30 Major findings Cadmium is associated with shivs in mirna and mrna signaling in heart cells mirnas are associated with known metal- responsive factors Many of the transcripts encode proteins with key func4onality in the cell 30

31 Trans- disciplinary areas: Environmental metals and epigene4cs Exposure Science Epidemiology Toxicology 31

32 mirna expression in the cervix and the associa4on with preterm birth and gesta4onal age

Preterm Birth Annually in US over 450,000 infants born before 37 weeks gesta4on (~12%) Risk factors for preterm birth Race Smoking Poverty Mul4ple births Short

33 Preterm Birth Annually in US over 450,000 infants born before 37 weeks gesta4on (~12%) Risk factors for preterm birth Race Smoking Poverty Mul4ple births Short cervix Bacterial vaginosis (BV) Mechanisms for spontaneous preterm birth remain poorly understood. Accessed July 16, 2013: from google image search premature infant 33

34 Epigene4cs and Preterm Most epigene4c studies Placental insufficiency/ preeclampsia Poor fetal growth Blood DNA, placenta Accessed July 16, 2013: from google image search placenta 34

Epigene4cs and Preterm Most epigene4c studies Placental insufficiency/ preeclampsia Poor fetal growth Blood DNA, placenta Spontaneous preterm

35 Epigene4cs and Preterm Most epigene4c studies Placental insufficiency/ preeclampsia Poor fetal growth Blood DNA, placenta Spontaneous preterm Cervical incompetence Preterm labor Premature rupture of membranes Cervical samples Accessed July 16, 2013: from google image search placenta 35

36 mirnas involved in pregnancy and labor mir- 200 family regulates uterine quiescence and contrac4lity (Renthal et al. 2010) mir- 371 family, C14MC, C19MC placenta- specific pregnancy- associated mirna (reviewed by Morales et al. 2013). Control mirna dele4on mirna are vital for normal embryonic development (Huang et al., 2010) 36

37 Hypothesis (i): Changes in mirna expression in the cervix are associated with the length of gesta4on Hypothesis (ii): Environmental exposures alter mirna expression in the cervix 37

38 Aim 1: mirna à Gesta9onal age Exposure Smoking Blood Pb BPA Mercury Changes in gene expression Altered cell signaling and func9on Preterm Birth (Ahir et al., 2013) 38

39 Methods Birth Cohort Mexico City, (ELEMENT/ PROGRESS) Enrolled during 2 nd trimester 1054 mother- infant pairs Approached final 100 for consent for a mid- pregnancy cervical swab 80 agreed 39

40 Methods Cervical swab collected during the 2 nd trimester (n=80) RNA extracted from 60 cervical samples Nanostring ncounter system to assess mirna expression Data were background- corrected using the average of nega4ve controls. Probes that were detected in 60% of the samples were included and the data were quan4le normalized. 40

41 Demographics for nested cohort n (%) Maternal age (years) ave ± std (range) 27.9 ± 5.7 (18-40) Educa9on < 12 years 19 (32) 12 years 24 (40) > 12 years 17 (28) Pre- pregnancy BMI (kg/m2) Normal BMI < (50) Overweight 25 - < (32) Obese >= (18) Smoke in home Yes 18 (30) No 42 (70) Gesta9onal age* (weeks) ave ± std Gesta9onal age* (days) ave ± std 38.2 ± 1.4 (34-42) 271 ± 10 ( ) Blood Pb (ug/dl) ave ± std (range) 2.8 ± 1.6 ( ) Toenail Hg (ug/g) ave ± std (range) 0.17 ± 0.09 ( ) 41

42 Regression models Associa4ons between 84 mirna expression levels and gesta4onal age (days). A priori covariates: Maternal age, pre- pregnancy BMI (normal [ref], overweight, obese), educa4on status (<HS, HS [ref], >HS), ETS, inflamma4on on Pap smear Associa4ons between environmental exposures (blood lead, BPA, and toenail Hg) with mirna expression in the cervix during pregnancy. 42

43 Trends in mir expression with gesta4onal age 43

44 Predic4on of downstream targets: mirna target hundreds of mrna

45 Network analysis Enriched for gene expression, cellular development, and skeletal and muscular system development and func4on Inflamma4on Hormone signaling p=1x

46 Conclusions Iden4fied dis4nct sets of up- and down- regulated mirna in the cervix associated with gesta4onal age Limita4ons: mirna plaorm, 4me of sample collec4on during pregnancy, ethnically homogenous popula4on, confirma4on of downstream targets Prospec4vely enrolled, ideal target 4ssue, largest n to- date 46

47 Future direc4ons Confirm downstream mrna target expression Pathophysiology of the cervix à preterm birth Some of our top mirs upregulated with maternal lead & mercury exposure Therapies for interven4on: mirnas as poten4al biomarker, antagomirs as treatment? mir Antagomir 47

Poten4al epigene4c mechanism by which Cd may alter cardiomyocyte func4on Poten4al mirnas

48 Public health relevance and conclusions Poten4al rela4onships between metals, mirna, and two leading causes of infant mortality Cd alters mirnas involved in heart development and func4on Poten4al epigene4c mechanism by which Cd may alter cardiomyocyte func4on Poten4al mirnas involved in pathophysiology of the cervix that may lead to PTB Health Effects Exposure Mechanism 48

49 Public health relevance and conclusions Metals are a widespread toxicants Early- life exposures can alter the epigenome Poten4ally preventable environmentally- mediated diseases 49

50 Cervix mirna Team Harvard Andrea Baccarelli Heather Burris* Allan Just Valeria Mo a Joel Schwartz Mount Sinai Robert Wright Katherine Svensson Mexico Adriana Mercado-Garcia Mara Tellez-Rojo R01ES013744; R01ES020268; R01ES014930; R01ES

51 Cd- cardiomyocyte Team Fry Laboratory Rebecca Fry Ka4e Bailey Julia Rager Paul Ray Daniel Rojas Lisa Smeester 51

52 Thank you! A. Arsenic Average arsenic conc (ppb)

RNA- seq Introduc1on. Promises and pi7alls

RNA- seq Introduc1on. Promises and pi7alls RNA- seq Introduc1on Promises and pi7alls DNA is the same in all cells but which RNAs that is present is different in all cells There is a wide variety of different func1onal RNAs Which RNAs (and some1mes