SYSTEMS BIOLOGY AND TOXIC METALS: LINKING BIOLOGICAL PATHWAYS AND LONG TERM HUMAN HEALTH EFFECTS Department of Environmental Sciences and Engineering, Gillings School of Global Public Health, University of North Carolina, Chapel Hill, NC, USA

Prevent detrimental health effects from exposure Explore biological effects of exposure to environmental agents: Fry Lab Mission Understand impact on human health: Molecular basis for disease Accurately detect exposure Biomarkers of population exposure, biomarkers of disease state Predict inter-individual differences in susceptibility to disease

A global poison: iAs contamination is affecting individuals around the world iAs continues to poison the drinking water of tens of millions of people around the world

A global poison: iAs contamination is affecting individuals around the world iAs continues to poison the drinking water of tens of millions of people around the world Southeast Asia alone 40 million exposed to levels above 50 ppb Southeast Asia alone 40 million exposed to levels above 50 ppb

iAs continues to poison the drinking water of tens of millions of people around the world Raise awareness of areas of concern

 Abstract #477: AP Sanders: Association between metals in private wells and birth defects More than 2.3 million people in North Carolina use water from private, unregulated wells ,000 people >63,000 wells over 10 yrs 1436 wells >10 ppb Hundreds > 50 ppb Max=800 ppb >63,000 wells over 10 yrs 1436 wells >10 ppb Hundreds > 50 ppb Max=800 ppb Sanders et al. Environ Int 2012

Toxic metals are detectable in pregnant women in North Carolina Lead: 1 exceeds CDC pregnant women advisory (5 ug/dL) Mercury: 5 exceed pregnancy level of concern (3.5 ug/L)

Health effects of arsenic: cancer and non cancer endpoints  Cancer (Group 1 IARC)  Liver, lung, bladder, kidney, prostate  Non-cancer  peripheral vascular disease  cardiovascular disease (e.g. atherosclerosis)  neurological effects  birth outcomes  diabetes

Health effects of arsenic: cancer and non cancer endpoints  Cancer (Group 1 IARC)  Liver, lung, bladder, kidney, prostate  Non-cancer  peripheral vascular disease  cardiovascular disease (e.g. atherosclerosis)  neurological effects  birth outcomes  diabetes

Unraveling the complex mode of action of iAs  iAs is not a point mutagen  iAs is generally negative in standard animal carcinogenesis studies  Research supports complex mode of action Arsenic- induced disease Generation of Oxidative Stress Altered Cell Signaling Cascades Interference with DNA repair Enzyme inhibition Chromosomal Aberrations Epigenetic Alterations

in utero exposure to iAs in rodents- alarming findings CD1 mice, exposed to 85 ppm iAs increase in hepatocellular carcinomas Gene expression changes in livers of offspring exposed to arsenic in utero when reach adulthood DNA methylation changes in target tissues-(ER- α showed hypomethylation) CD1 mice, exposed to 85 ppm iAs increase in hepatocellular carcinomas Gene expression changes in livers of offspring exposed to arsenic in utero when reach adulthood DNA methylation changes in target tissues-(ER- α showed hypomethylation) Waalkes, M. P. et al Toxicol Appl Pharmacol, (2004). Waalkes, M. P., et al, Journal of the National Cancer Institute, (2004). Xie, Y., et al, Toxicology, (2007). In utero exposure is associated with adult onset disease

Prenatal and early life iAs exposure in humans and mortality Increased mortality from bladder, kidney, liver and lung cancer from prenatal and early childhood exposures (Smith et al 2012, Liaw et al., 2008; Smith et al., 2006). Prenatal exposure in humans and adult disease: supporting epigenetic modifications Cancer Non-Cancer

What are biological mechanisms underlying the long-term health effects associated with early life arsenic exposure?

Establishing a prospective maternal-child cohort: Gómez Palacio, Mexico Gómez Palacio, Mexico García Vargas Study launched in 2010 (ONES NIEHS) Concerns over iAs in water (LM Del Razo) Research network (M Styblo) Prenatal exposure to iAs has not been assessed >200 mother-baby pairs recruited Study launched in 2010 (ONES NIEHS) Concerns over iAs in water (LM Del Razo) Research network (M Styblo) Prenatal exposure to iAs has not been assessed >200 mother-baby pairs recruited

Various endpoints as biomarkers of exposure: Integrated view of systems-wide effects of iAs Inform mechanism of disease Collection and isolation of samples for protein, mRNA, DNA assessment

Biomarkers of Exposure to Arsenic The BEAR Study Inform mechanism of disease

Pregnant women are exposed to high levels of iAs through drinking water 53% 28% % Collected urine during third trimester of pregnancy and drinking water from the home

Pregnant women are exposed to high levels of iAs through drinking water 53% 28% N=107 (53%) exposed to >10 ppb N=56 (28%) exposed to >25 ppb Range in water <1 ppb to 240 ppb UAs to WAs p<0.01 Collected urine during third trimester of pregnancy and drinking water from the home %

Pregnant women are exposed to high levels of iAs through drinking water 53% 28% N=107 (53%) exposed to >10 ppb N=56 (28%) exposed to >25 ppb Range in water <1 ppb to 240 ppb Collected urine during third trimester of pregnancy and drinking water from the home % Abstract #473: JE Laine: Prenatal exposure to inorganic arsenic Pregnant women are being exposed to elevated levels of iAs in Mexico

National Academy of Sciences' 1999 risk estimates Arsenic Level in Tap Water (ppb) Approximate Total Cancer Risk 0.5 ppb1 in 10,000 1 ppb1 in 5,000 3 ppb1 in 1,667 4 ppb1 in 1,250 5 ppb1 in 1, ppb1 in 500 (50% in BEAR) 20 ppb1 in ppb1 in 200 (30% in BEAR) 50 ppb1 in 100 lifetime risks of dying of cancer from arsenic in tap water

National Academy of Sciences' 1999 risk estimates Arsenic Level in Tap Water (ppb) Approximate Total Cancer Risk 0.5 ppb1 in 10,000 1 ppb1 in 5,000 3 ppb1 in 1,667 4 ppb1 in 1,250 5 ppb1 in 1, ppb1 in 500 (50% in BEAR) 20 ppb1 in ppb1 in 200 (30% in BEAR) 50 ppb1 in 100 lifetime risks of dying of cancer from arsenic in tap water

National Academy of Sciences' 1999 risk estimates Arsenic Level in Tap Water (ppb) Approximate Total Cancer Risk 0.5 ppb1 in 10,000 1 ppb1 in 5,000 3 ppb1 in 1,667 4 ppb1 in 1,250 5 ppb1 in 1, ppb1 in 500 (50% in BEAR) 20 ppb1 in ppb1 in 200 (30% in BEAR) 50 ppb1 in 100 lifetime risks of dying of cancer from arsenic in tap water

Are there proteins with altered expression levels in the cord blood of babies who experienced prenatal arsenic exposure? Concerns for the developing baby

Subcohort of 50 newborns selected from BEAR: serum from cord blood analyzed 121 ppb 11 ppb Newborns with low prenatal iAs (wAs <5ppb) Newborns with high prenatal iAs (wAs >100ppb)

Proteins assessed in cord blood using proteomics assay >500 proteins assessed Cytokines Chemokines Growth factors Angiogenic factors Soluble receptors >500 proteins assessed Cytokines Chemokines Growth factors Angiogenic factors Soluble receptors proteins are biotinylated at primary amines protein-specific antibodies are on array For each protein, across the 50 samples, regression analysis of urinary iAs as a continuous variable related to protein expression, controlling for potential confounders

31 proteins with altered expression associated with prenatal iAs levels 23 with increased expression as iAs increases 8 with decreased expression as iAs increases Fibrobast growth factor 20 Interleukin 23 Intensity units

17 of 31 in highly significant network p< Increased expression Decreased expression Proteins interact in a common pathway

17 of 31 in highly significant network p< Proteins interact in a common pathway ERK 1/2 signaling pathway: Extracellular-signal-regulated kinase ERK 1/2 signaling pathway: Extracellular-signal-regulated kinase Increased expression Decreased expression Mitogen-activated protein kinase pathway linked to cellular growth and proliferation Implicated in carcinogenesis, key mediator of inflammatory responses pathway modulated by arsenic

17 of 31 in highly significant network p< Proteins interact in a common pathway: known links to iAs Increased expression Decreased expression Mitogen-activated protein kinases linked to cellular growth and proliferation Implicated in carcinogenesis, key mediator of inflammatory responses pathway modulated by arsenic EGFR: Upregulated in serum in humans exposed to iAs ERK2: Activated by iAsIII, MMAIII, DMAIII in vitro MIF and EGFR: Upregulated by MMAIII in vitro TIMP2: Upregulated by arsenite in the mouse liver

Macrophage inhibitory factor Interleukin 27 receptor subunit alpha Interleukin 1 receptor like 2 Histidine-rich glycoprotein SMAD family member 4/5 Epiregulin Proteins are pro-inflammatory Environ Health Perspect Feb;119(2): Arsenic-associated oxidative stress, inflammation, and immune disruption in human placenta and cord blood. Ahmed S, et al. International Centre for Diarrhoeal Disease Research, Bangladesh, Dhaka, Bangladesh, 18 cytokines

Macrophage inhibitory factor Interleukin 27 receptor subunit alpha Interleukin 1 receptor like 2 Histidine-rich glycoprotein SMAD family member 4/5 Epiregulin Proteins play a role in carcinogenesis Environ Health Perspect Feb;119(2): Arsenic-associated oxidative stress, inflammation, and immune disruption in human placenta and cord blood. Ahmed S, et al. International Centre for Diarrhoeal Disease Research, Bangladesh, Dhaka, Bangladesh, 18 cytokines Lung/liver Liver

Proteins play a role in carcinogenesis Macrophage inhibitory factor Epidermal growth factor receptor Epiregulin Matrix metallopeptidase 13 Mothers against decapentaplegic homolog 4 Increased expression in lung and liver tumors (MIF, EGFR) Metastasis and invasion in tumors (MMP13, CXCL16, ICAM) Prognostic indicators for hepatocellular carcinoma (SMAD 4) Increased expression in lung and liver tumors (MIF, EGFR) Metastasis and invasion in tumors (MMP13, CXCL16, ICAM) Prognostic indicators for hepatocellular carcinoma (SMAD 4)

In utero exposure to iAs in Thailand: gene expression Fry et al., 2007 Generating a systems level view of the effects of iAs

In utero exposure to iAs in Thailand: gene expression Fry et al., 2007 Proteomic and genomic signaling enriched for inflammation and immune response Proteomic and genomic signaling enriched for inflammation and immune response Generating a systems level view of the effects of iAs

Changes in protein expression in cohort in Mexico Changes in protein expression in cohort in Mexico Generating a systems level view of the effects of iAs

Changes in protein expression in cohort in Mexico Changes in protein expression in cohort in Mexico Changes in gene expression in cohort in Thailand Changes in gene expression in cohort in Thailand Generating a systems level view of the effects of iAs

Changes in protein expression in cohort in Mexico Changes in protein expression in cohort in Mexico Changes in gene expression in cohort in Thailand Changes in gene expression in cohort in Thailand DNA methylation? Generating a systems level view of the effects of iAs

DNA methylation: a key component of the epigenetic machinery SAM

DNA methylation at promoter regions can impede target gene expression Promoter Methyl CpG TF Methyl CpG X Target gene Promoter TF Target gene Target gene silenced Target gene expressed

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Putative mechanisms for arsenic- induced changes to DNA methylation SAM

Putative mechanisms for arsenic- induced changes to DNA methylation SAM Zhou et al 1997 Reichard et al 2007

Are there iAs-associated differences in DNA methylation of the genes encoding the protein biomarkers?

Extensive differences in gene-specific DNA methylation patterns in adults exposed to iAs ~200 genes Smeester et al. 2011

Proteomic changes correspond with DNA methylation profiles  450,000 methylation sites /single- nucleotide resolution  99% of RefSeq genes  48 cord blood samples, analyzed for DNA methylation associated with UAs

Proteomic changes correspond with DNA methylation profiles (n=10/31) CXCL16 PECAM1 TIMP2 ICAM3 IL27RA NCAM1 CCL5 SMAD5 EGFR NRG3  450,000 methylation sites /single- nucleotide resolution  99% of RefSeq genes  48 cord blood samples, analyzed for DNA methylation associated with UAs

Proteomic changes correspond with DNA methylation profiles (n=10/31) CXCL16 PECAM1 TIMP2 ICAM3 IL27RA NCAM1 CCL5 SMAD5 EGFR NRG3 Some of proteomic response linked to inflammation and carcinogenesis in newborn cord blood may be mediated by DNA methylation Some of proteomic response linked to inflammation and carcinogenesis in newborn cord blood may be mediated by DNA methylation  450,000 methylation sites /single- nucleotide resolution  99% of RefSeq genes  48 cord blood samples, analyzed for DNA methylation associated with UAs

Summary  Arsenic continues to poison the water of individuals around the globe, including North Carolina and Mexico  Proteomic shifts of the ERK pathway associated with prenatal arsenic exposure in newborns in Mexico  Some overlap at the level of DNA methylation between genes altered by iAs exposure

UNC-Chapel Hill Fry Lab Bhavesh Ahir, Ph.D., Kathryn Bailey, Ph.D. Daniel Rojas, Julia Rager Alison Sanders, Jessica Laine Lisa Smeester Collaborators Zuzana Drobná, Ph.D., Xiaojun Guan, Ph.D. Hemant Kelkar, Ph.D., Miroslav Stýblo, Ph.D. UNC-Chapel Hill Fry Lab Bhavesh Ahir, Ph.D., Kathryn Bailey, Ph.D. Daniel Rojas, Julia Rager Alison Sanders, Jessica Laine Lisa Smeester Collaborators Zuzana Drobná, Ph.D., Xiaojun Guan, Ph.D. Hemant Kelkar, Ph.D., Miroslav Stýblo, Ph.D. Juarez University, Durango State, Mexico Gonzalo G. García Vargas M.D., Ph.D. Juarez University, Durango State, Mexico Gonzalo G. García Vargas M.D., Ph.D. Funding NIEHS (ONES): R01ES NIEHS CEHS UNC: P30ES NIEHS Superfund: P42 ES Funding NIEHS (ONES): R01ES NIEHS CEHS UNC: P30ES NIEHS Superfund: P42 ES005948