systems biology and toxic metals: linking biological pathways and long term human health effects...
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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
iAs continues to poison the drinking water of tens of millions of people around the world
Southeast Asia alone40 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
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, 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
Inform mechanism of diseaseCollection and isolation of samples for protein, mRNA, DNA assessment
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 ppbN=56 (28%) exposed to >25 ppbRange in water <1 ppb to 240 ppbUAs 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 ppbN=56 (28%) exposed to >25 ppbRange 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 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
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
nsi
ty u
nits
Inte
nsi
ty u
nits
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
Proteins interact in a common pathway
ERK 1/2 signaling pathway: Extracellular-signal-
regulated kinase
Increased expression
Decreased expressionMitogen-activated protein kinase pathway linked to cellular growth and proliferationImplicated in carcinogenesis, key mediator of inflammatory responsespathway modulated by arsenic
17 of 31 in highly significant network p< 10-41
Proteins interact in a common pathway: known links to iAs
Increased expression
Decreased expressionMitogen-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
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. 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
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)
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
Generating a systems level view of the effects of iAs
Changes in protein expressionin cohort in Mexico
Generating a systems level view of the effects of iAs
Changes in protein expressionin cohort in Mexico
Changes in gene expressionin cohort in Thailand
Generating a systems level view of the effects of iAs
Changes in protein expressionin cohort in Mexico
Changes in gene expressionin cohort in Thailand
DNA methylation?
Generating a systems level view of the effects of iAs
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
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,
analyzed for DNA methylation associated with UAs
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,
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 RagerAlison 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): R01ES019315NIEHS CEHS UNC: P30ES010126 NIEHS Superfund: P42 ES005948