Transcript Gene Expression Signatures as Bioindicators of Exposure and

Gene Expression Signatures as
Bioindicators of Exposure and
Health Effects Related to
Environmental Contaminants
Watershed Ecosystem and Human Health Risk
Assessment Process
Ecosystem & Human Health
Risk Assessment
•Risk Assessor /
Risk Manager
Dialogue
•Identify Goals and Assessment Endpoints
•Preparing Conceptual Model
•Developing Analysis
Watershed
Modeling
Stressor
Exposure
Analysis
Receptor
Response
Community
Response
•Resource Use & Exposure
Communicating Results to
Risk Managers and Stakeholders
Data Sources:
•DNR
•Public Health
•County
•MI CGI
•Research Reports
Limitations to Environmental
Risk Assessment
• Ecological risk assessment
– Difficulties in assessing exposure and related health
effects in the field
• Traditionally relies on population distribution and
biodiversity analysis
• Time consuming, expensive, lacks precision
• Human health risk assessment
– Extremely difficult to isolate environmental causes of clinical
health effects
– Shortcomings of epidemiology studies due to diverse lifestyles
Biotechnology: Molecular Based
Eco Risk Assessment (WMU)
• In the laboratory, define gene expression
patterns in healthy versus ill animals
exposed to environmental contaminants.
• Using biotech tools, measure gene
expression patterns in wildlife (or humans)
exposed to contaminants.
• Gene expression predicts exposure and
cryptic health effects.
• Use data to determine cleanup priorities.
Gene Expression as Diagnostics
for Health and Disease
• 30,000 genes are expressed in humans.
• Many are expressed during good health
(normal physiology).
• During illness, some are over expressed
(immune system genes, beta amyloid gene
in Alzheimer’s disease), while some are
under expressed (dopamine synthesis genes
in Parkinson’s disease).
Gene Expression
• Genes sequences
code for
• Complementary
mRNA sequences
code for
• Amino acid
sequences that are
• Proteins that
underlie living
processes.
Initial Proof of Concept Studies
• Load tadpole and fish tissues with PCB
concentrations found in Kalamazoo River wildlife
– Short approach: use DMSO to carry PCBs into
animals to desired concentration (1 day to 1
week exposure)
– Long approach: feed PCB laced food over
weeks to appropriate tissue levels
• Observe behavior, morphology, mortality, gene
expression signatures
Bioindicator Genes
• Induction of Apoptosis
– CPP32ß (caspase-3)
– ICE (interleukin-1
converting enzyme)
• Endocrine Control
– Retinoic acid receptor
– Thyroxine receptor
– POMC (proopiomelanocortin)
• Neurological Function
– D2 dopamine receptor
– Nerve growth factor (NGF)
• Control of cell cycle/Cell
structure/Metabolic
control
– p53
– ß-actin
– GAPDH
• Response to xenobiotics
– p450
• Multi-functional
– p53
– ICE
1000
900
120
A
110
100
800
700
§-actin
N GF
600
survival
90
**
80
**
**
70
**
500
60
50
400
40
300
30
200
20
100
10
0
0
c ontrol
5
50
250
500
Aroclor 125 4 (ppb)
expression/Control
Gene expression/Control
%
% Gene
CPP32ß
ICE
POMC
p53
DMSO
1200
120
110
1000
100
90
GAPDH
800
***
IC E
80
**
POMC
70
p53
600
**
survi val
***
**
60
***
50
*
400
40
30
200
20
10
0
0
control
DMSO
5
50
Aroclor 1254 (ppb)
250
500
% Survival/DMSO
% Survival/DMSO
–
–
–
–
% Survival/DMSO
• Exposure to low levels of
Aroclor 1254 (5 and 50 ppb)
increased gene expression
– NGF
– ß-actin
% Gene expression/Control
Gene Expression in 18 Day Old
Tadpoles: Bioindicators of Exposure
250
120
B
110
100
200
150
90
ß- actin
NGF
survival
80
**
70
60
100
*
*
**
**
50
*
**
40
30
50
20
10
0
0
control
DMSO
300
400
Aroclor 1254 (ppb)
500
700
% Survival/DMSO
• Decreased gene expression
at high doses (700 ppb)
correlated with decreased
survival and the onset of
adverse health effects
– NGF
– ß-actin
• Decreases in gene
expression occurred in
tadpoles exposed to 300
ppb and greater
% Gene expression/Control
Decreases in Gene Expression Are
Predictive Bioindicators
Gene Expression Signatures as Bioindicators of Exposure to
PCBs and Related Health Effects in Developing
Xenopus Frogs and carp, Cyprinus carpio
Project Coordinator: Charles F. Ide, Ph.D. Professor of Biological Sciences and
Director, Environmental Institute
Jay C. Means, Ph.D. Professor of Environmental Chemistry and Toxicology,
Associate Director, Environmental Institute
Co-Investigator: Anna M. Jelaso, Ph.D. Assistant Professor, Environmental
Institute
Co-Investigator: Marla A. Fisher, Ph.D. Postdoctoral Researcher, Department
of Environmental and Molecular Toxicology, NCSU
Dr. Bharti Katbamna, Professor of Speech Pathology and Audiology
Libby Lehigh-Shirey, Ron Celestine - Graduate Students
New Frog Gene Expression Work
• DMSO assisted Aroclor 1254 exposure studies (2
day exposures) showed that tissue levels <50 ppm
showed increases in gene expression, no external
health effects; levels >100 ppm showed decreases
in gene expression and overt health effects and
mortality
• Need to determine if tissue levels < 100 ppm
produce cryptic health effects in real word
exposure (dietary) setting (can gene expression
analysis reveal cryptic health effects that will
ultimately alter fitness)
New Gene Expression Work
• Pilot work shows frogs exposed to low levels of
PCBs (50ppb, DMSO) or through diet (12, 24
ppm) showed slow metamorphic rates
• Important for frog population fitness due seasonal
limitations on frog lifestyle
• Determine gene expression changes underlying
PCB induced changes in metamorphic rate
Origin
Function
CRF (corticotropin
releasing factor)
Hypothalamus
Stimulates ACTH release;
Stimulates TSH release in
amphibians
POMC (proopiomelanocortin)
Pituitary
Precursor to ACTH and
Melanin

TSH (thyroid
stimulating
hormone)
Pituitary
Stimulates TH production in
thyroid gland

TR-beta (thyroid
hormone receptor)
Target tissue
Forms heterodimer with
RXR; Binding of TH

D2 (Type II
Deiodinase)
Target tissue;
mainly the brain
Converts T4 into T3
D3 (Type III
Deiodinase)
Target tissue
Converts T4 and T3 into rT3
and T2, biologically inactive
forms
Gene
Change in
gene
expression



New Gene Expression Work
• Perform chronic 60 and 90 day dietary
exposures (0, 12, 50, 100, 200 ppm Aroclor
1254)
• Analyze previous gene battery including
thyroid system genes
• Analyze new endocrine disruption gene
battery (wnt4a, sox3, ER, AR, CYP17,
CYP19)
New Gene Expression Work
• PCB exposure in utero reduces low frequency hearing in
humans
• Established in mammalian models, but not relevant
ecosystem models
• Expose developing frogs to PCBs (dietary) and assay
development of auditory function using Auditory Brainstem
Response Methods
• Correlate with gene expression analysis for genes involved
in development of vestibulo-auditory brain circuits (NGF,
CX 43, CX 31)
• Will establish a model system for assaying cryptic
contaminant induced health effects in wildlife, and will
provide basic data regarding genes controlling auditory
system development
New Gene Expression Work
• High atrazine levels present in the St. Joseph River watershed
and in Lake Michigan
• Controversial morphology and histology based studies claim
that atrazine feminizes male frogs at <environmental levels
• Settle controversy by exposing frogs to atrazine (0.1, 25 ppb)
and measuring gene expression for genes that determine
sexual phenotype (wnt4a for males, sox3 for females; also
CYP19 and CYP17 aromatase, and for ER and AR) in treated
and untreated animals
• Also, pilot data shows that low level atrazine treatment speeds
up metamorphosis, so measure expression of thyroid system
genes
• Should establish a molecular basis for endocrine disruption
effects of atrazine at environmental levels
New Gene Expression Work - Carp
• Carp are the most contaminated fish in the Kalamazoo
River (up to 164 ppm PCBs)
• Exposed carp in the laboratory through diet (12 ppm
Aroclor 1242) for 1, 2, 3, 4 months
• Gene expression analysis showed upregulation P4501A
(bioindicator of exposure)
• Gene expression changes also present in carp caught from
contaminated river sites versus cleaner sites
• Increased liver (hepatopancreas) macrophage aggregates in
carp caught from contaminated river sites versus cleaner
sites
Kalamazoo River: Superfund Site Due to
PCB Contamination
PCB Levels in Carp Tissues
Muscle PCBs
(ug/g)
Laboratory Exposed Carp
Days Fed PCBs (A1242)
Kalamazoo River Carp
PCB Induced p4501A1 Gene Expression in
Carp
P450 1A1 mRNA
(ng/ul)
Laboratory Exposed Carp
Days Fed PCBs (A1242)
Kalamazoo River Carp
Histopathology: Kalamazoo River Carp
Macrophage Aggregates
•Hepatocyte and exocrine pancreas
macrophage aggregates
•Stain: H&E
Example macrophage aggregate showing
lipofuscin, hemosiderin, melanin
Gamori’s Prussian Blue, no counterstain
Histopathology Quantification: PCB
Sites Increased MA Area and MA #
Macrophage Aggregate #/Area
significantly increased in carp from
PCB vs reference sites (ANOVA,
p=0.0453; n=17 reference, n=10 PCB)
Significantly different among sites
(p=.0079)
Lake Allegan higher than
Trowbridge, Ceresco, Morrow
4.5
% MA Area (mm2)
% Macrophage Aggregate Area
significantly increased in carp from
PCB vs reference sites (ANOVA
p=0.026; n=17 reference, n=10 PCB)
Despite carp from PCB sites are
younger/smaller
Increase not from age/size
4
3.5
3
2.5
2
1.5
1
0.5
0
Control
PCB
Meaning of Increased Macrophage
Aggregates in PCB exposed carp
• Carp hepatopancreas macrophage aggregates
consist primarily of lipofuscin
• Increase in MA densities in carp hp from PCB
sites  Increase in lipofuscin  Increased need
for lipid clearance and storage in MAs in PCB
exposed carp
Correlation of hepatopancreas MA
Densities with hepatopancreas PCBs
Correlation
Coefficient
Control Sites
Ceresco
Morrow
PCB Sites
Lake Allegan
Trowbridge
% MA Area
(mm2)
MA #/ Area
(mm2)
MA size
(mm2)
r
p
r
p
-0.19
0.61
0.27
0.51
-0.16
0.67
0.06
0.88
-0.38
0.31
0.15
0.72
r
p
r
p
0.81
0.09
0.60
0.29
0.91
0.03*
0.60
0.28
-0.03
0.96
0.58
0.31
PCBs are major contaminant in Kalamazoo River
In general, carp from reference sites: low r, high p
Carp from PCB sites: high r, low p
Lake Allegan carp: MA# significantly correlated with PCB levels
Suggests distinct carp population at this site (Kalamazoo RiverDams)
Suggests population responding to PCBs (vs stunted growth?)
Carp Model
• Carp contaminates with high levels of PCBs are found
in the Kalamazoo River Superfund Site
• CYP1A mRNA, an indicator of exposure to PCBs, was
elevated in carp liver from PCB contaminated sites
compared to upstream reference sites
• Macrophage Aggregate densities were elevated in carp
liver from PCB contaminated sites compared to
reference sites
• Molecular data plus histopathology data suggest carp
in PCB exposed sites are responding to exposure
• Data will be viewed in light of claims by PRP
scientists that contaminated organisms in the river are
in good health