Systems biology and toxic metals: Linking biological pathways and long term human health effects

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 exposureBiomarkers 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


Southeast Asia alone40 million

exposed to levels above 50 ppb


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

2009

75,000 people>63,000 wells over 10 yrs1436 wells >10 ppbHundreds > 50 ppbMax=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

repairEnzyme inhibition

Chromosomal

Aberrations

Epigenetic Alterations

in utero exposure to iAs in rodents-alarming findings

CD1 mice, exposed to 85 ppm iAsincrease 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).

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, MexicoGarcía 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

Inform mechanism of diseaseCollection 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


53%

28%

N=107 (53%) exposed to >10 ppbN=56 (28%) exposed to >25 ppbRange in water <1 ppb to 240 ppbUAs to WAs p<0.01


%


53%

28%

N=107 (53%) exposed to >10 ppbN=56 (28%) exposed to >25 ppbRange in water <1 ppb to 240 ppb


%

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 ppb 1 in 10,0001 ppb 1 in 5,0003 ppb 1 in 1,6674 ppb 1 in 1,2505 ppb 1 in 1,00010 ppb 1 in 500 (50% in BEAR)20 ppb 1 in 25025 ppb 1 in 200 (30% in BEAR)50 ppb 1 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 assessedCytokines

ChemokinesGrowth factors

Angiogenic factorsSoluble 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 expressionas iAs increases

8 with decreased expressionas iAs increases

Fibrobast growth factor 20Interleukin 23

Inte

nsity

uni

ts

Inte

nsity

uni

ts

17 of 31 in highly significant network p< 10-41

Increased expressionDecreased expression

Proteins interact in a common pathway


Proteins interact in a common pathway

ERK 1/2 signaling pathway: Extracellular-signal-

regulated kinase


Mitogen-activated protein kinase pathway linked to cellular growth and proliferationImplicated in carcinogenesis, key mediator of inflammatory responsespathway modulated by arsenic


Proteins interact in a common pathway: known links to iAs


Mitogen-activated protein kinases linked to cellular growth and proliferationImplicated in carcinogenesis, key mediator of inflammatory responsespathway 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. 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


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. 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

Lung/liver

Liver

Proteins play a role in carcinogenesis


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)

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



Changes in protein expressionin cohort in Mexico



Changes in gene expressionin cohort in Thailand



Changes in gene expressionin cohort in Thailand

DNA methylation?


DNA methylation: a key component of the epigenetic machinery

SAM

SAM

DNA methylation at promoter regions can impede target gene expression

Promoter

MethylCpG

TF

MethylCpG

X Target gene

Promoter

TF

Target gene

Target gene silenced

Target gene expressed

42

43

Putative mechanisms for arsenic-induced changes to DNA methylation

SAM

SAM

Putative mechanisms for arsenic-induced changes to DNA methylation

SAM

Zhou et al 1997Reichard et al 2007

Are there iAs-associated differences inDNA 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)

CXCL16PECAM1TIMP2ICAM3IL27RANCAM1CCL5SMAD5EGFRNRG3

450,000 methylation sites /single-nucleotide resolution

99% of RefSeq genes 48 cord blood samples,


Proteomic changes correspond with DNA methylation profiles (n=10/31)

CXCL16PECAM1TIMP2ICAM3IL27RANCAM1CCL5SMAD5EGFRNRG3

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,


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 LaineLisa 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.

FundingNIEHS (ONES): R01ES019315

NIEHS CEHS UNC: P30ES010126 NIEHS Superfund: P42 ES005948

Documents

Systems biology and toxic metals: Linking biological pathways and long term human health effects