Transcript Dr. Fry`s presentation

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
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
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
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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)
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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