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 Fry Lab Mission Explore biological effects of exposure to environmental agents: 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 Prevent detrimental health effects from exposure 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 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 iAs continues to poison the drinking water of tens of millions of people around the world Raise awareness of areas of concern More than 2.3 million people in North Carolina use water from private, unregulated wells 2009 75,000 people >63,000 wells over 10 yrs 1436 wells >10 ppb Hundreds > 50 ppb Max=800 ppb Sanders et al. Environ Int 2012 Abstract #477: AP Sanders: Association between metals in private wells and birth defects Toxic metals are detectable in pregnant women in North Carolina Mercury: 5 exceed pregnancy level of concern (3.5 ug/L) Lead: 1 exceeds CDC pregnant women advisory (5 ug/dL) 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 Generation of Oxidative Stress Epigenetic Alterations Altered Cell Signaling Cascades Arsenicinduced disease Chromosomal Aberrations Enzyme inhibition Interference with DNA repair iAs is not a point mutagen iAs is generally negative in standard animal carcinogenesis studies Research supports complex mode of action in utero exposure to iAs in rodentsalarming findings In utero exposure is associated with adult onset disease 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, 198. 377-384 (2004). Waalkes, M. P., et al, Journal of the National Cancer Institute, 96. 466-474 (2004). Xie, Y., et al, Toxicology, 236. 7-15 (2007). Prenatal and early life iAs exposure in humans and mortality Cancer Non-Cancer 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 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 Various endpoints as biomarkers of exposure: Integrated view of systems-wide effects of iAs Collection and isolation of samples for protein, mRNA, DNA assessment Inform mechanism of disease 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% % 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 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 N=107 (53%) exposed to >10 ppb N=56 (28%) exposed to >25 ppb in water <1 ppb to 240 ppb inRange Mexico Pregnant women are being exposed to elevated levels of iAs 53% % 28% Collected urine during third trimester of pregnancy and drinking water from the home Abstract #473: JE Laine: Prenatal exposure to inorganic arsenic National Academy of Sciences' 1999 risk estimates Arsenic Level in Tap Water (ppb) Approximate Total Cancer Risk 0.5 ppb 1 in 10,000 1 ppb 1 in 5,000 3 ppb 1 in 1,667 4 ppb 1 in 1,250 5 ppb 1 in 1,000 10 ppb 1 in 500 (50% in BEAR) 20 ppb 1 in 250 25 ppb 1 in 200 (30% in BEAR) 50 ppb 1 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 ppb 1 in 10,000 1 ppb 1 in 5,000 3 ppb 1 in 1,667 4 ppb 1 in 1,250 5 ppb 1 in 1,000 10 ppb 1 in 500 (50% in BEAR) 20 ppb 1 in 250 25 ppb 1 in 200 (30% in BEAR) 50 ppb 1 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 ppb 1 in 10,000 1 ppb 1 in 5,000 3 ppb 1 in 1,667 4 ppb 1 in 1,250 5 ppb 1 in 1,000 10 ppb 1 in 500 (50% in BEAR) 20 ppb 1 in 250 25 ppb 1 in 200 (30% in BEAR) 50 ppb 1 in 100 lifetime risks of dying of cancer from arsenic in tap water Concerns for the developing baby Are there proteins with altered expression levels in the cord blood of babies who experienced prenatal arsenic exposure? 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 proteins are biotinylated at primary amines >500 proteins assessed Cytokines Chemokines Growth factors Angiogenic factors Soluble receptors 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 Fibrobast growth factor 20 Intensity units Intensity units Interleukin 23 8 with decreased expression as iAs increases 23 with increased expression as iAs increases Proteins interact in a common pathway 17 of 31 in highly significant network p< 10-41 Increased expression Decreased expression Proteins interact in a common pathway 17 of 31 in highly significant network p< 10-41 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 Proteins interact in a common pathway: known links to iAs 17 of 31 in highly significant network p< 10-41 ERK2: Activated by iAsIII, MMAIII, DMAIII in vitro MIF and EGFR: Upregulated by MMAIII in vitro TIMP2: Upregulated by arsenite in the mouse liver EGFR: Upregulated in serum in humans exposed 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 Proteins are pro-inflammatory Macrophage inhibitory factor Interleukin 1 receptor like 2 Histidine-rich glycoprotein Epiregulin SMAD family member 4/5 Interleukin 27 receptor subunit alpha Environ Health Perspect. 2011 Feb;119(2):258-64. 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 Proteins play a role in carcinogenesis Lung/liver Macrophage inhibitory factor Interleukin 1 receptor like 2 Histidine-rich glycoprotein Epiregulin Liver SMAD family member 4/5 Interleukin 27 receptor subunit alpha Environ Health Perspect. 2011 Feb;119(2):258-64. 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 Proteins play a role in carcinogenesis Macrophage inhibitory factor Increased expression in lung and liver tumors (MIF, EGFR) Epiregulin Epidermaland growth factor receptor Metastasis invasion in tumors (MMP13, CXCL16, ICAM) Matrix metallopeptidase 13 Prognostic indicators fordecapentaplegic hepatocellular carcinoma Mothers against homolog 4 (SMAD 4) Generating a systems level view of the effects of iAs In utero exposure to iAs in Thailand: gene expression Fry et al., 2007 Generating a systems level view of the effects of iAs Proteomic and genomic signaling enriched for inflammation and immune response In utero exposure to iAs in Thailand: gene expression Fry et al., 2007 Generating a systems level view of the effects of iAs Generating a systems level view of the effects of iAs 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 gene expression in cohort in Thailand Generating a systems level view of the effects of iAs DNA methylation? Changes in protein expression in cohort in Mexico Changes in gene expression in cohort in Thailand DNA methylation: a key component of the epigenetic machinery SAM SAM DNA methylation at promoter regions can impede target gene expression TF Target gene silenced Methyl Methyl CpG CpG Promoter X Target gene Target gene expressed TF Promoter Target gene 42 43 Putative mechanisms for arsenicinduced changes to DNA methylation SAM SAM Putative mechanisms for arsenicinduced 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 /singlenucleotide 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) 450,000 methylation sites /singlenucleotide resolution 99% of RefSeq genes 48 cord blood samples, analyzed for DNA methylation associated with UAs CXCL16 PECAM1 TIMP2 ICAM3 IL27RA NCAM1 CCL5 SMAD5 EGFR NRG3 Proteomic changes correspond with DNA methylation profiles (n=10/31) CXCL16 PECAM1 Some of proteomic response linked to TIMP2 inflammation and carcinogenesis in ICAM3 newborn cord blood may be mediated by IL27RA DNA methylation NCAM1 CCL5 450,000 methylation sites /singlenucleotide resolution SMAD5 99% of RefSeq genes EGFR 48 cord blood samples, analyzed for DNA methylation associated NRG3 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. Juarez University, Durango State, Mexico Gonzalo G. García Vargas M.D., Ph.D. Funding NIEHS (ONES): R01ES019315 NIEHS CEHS UNC: P30ES010126 NIEHS Superfund: P42 ES005948