The Adverse Outcome Pathway Approach to
Ecotoxicology and Its Possible Applicability to
Human Health Toxicology
Mixtures and Cumulative Risk Assessment: New Approaches:
Using the Latest Science and Thinking about Pathways
July 27–28, 2011
Ed Perkins, PhD
Senior Research Scientist
Environmental Laboratory
US Army Engineer Research and
Development Center
US Army Corps Engineers
Vicksburg, MS
email:
[email protected]
Raw
Material
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Lifetime
Article
Env.
Fate
Eco
health
Alt
species
Human
health
Rapid, inexpensive in vivo testing
Integrative
Dose effects
Metabolism
Developmental testing
• 24-96 well plate format
• Reproductive testing
• Daphnia 21 d reproductive test
• Fathead Minnow 21-d reproductive test
• Behavioral testing
• Exposure
• Relatively inexpensive
Systems toxicology and molecular analysis Pathway modeling
Rapid in vivo screening
Application of Research to
Levels of Organization Based
on Source to Outcome
Source
Community
Environmental
Contaminant
Population
Exposure
Individual
Key Event
Cellular Effects
Toxicity Pathway
Mode of Action
Adverse Outcome Pathway
Source to Outcome Pathway
Exposure
Uptake-Delivery to Target Tissues
Perturbation
Cellular response pathway
“Normal”
Biological
Function
Biologic
inputs
Adaptive
Responses
Early cellular
changes
Cell inury,
Inability to
regulate
4
Molecular
initiating event
Perturbed cellular
response pathway
Adverse outcome
relevant to
risk assessment
Toxicity Pathway
Adverse Outcome Pathway
Adverse
Outcomes
(e.g., mortality,
Reproductive
Impairment)
An Adverse Outcome Pathway (AOP) is a conceptual framework that portrays existing
knowledge concerning the linkage between a direct molecular initiating event and an
adverse outcome,
outcome, at a level of biological organization relevant to risk assessment.
(Ankley et al. 2010, Environ. Toxicol.
Toxicol. Chem., 29(3): 730730-741.)
Toxicant
Chemical
Properties
MacroMolecular
Interactions
Receptor/Ligand
Interaction
Cellular
Responses
Gene
activation
DNA Binding
Protein
production
Protein Oxidation
Altered
signaling
Organ
Responses
Organism
Responses
Population
Responses
Altered
physiology
Lethality
Structure
Impaired
Development
Recruitment
Disrupted
homeostasis
Altered tissue
development/
function
Impaired
Reproduction
Extinction
AOP and alternative animals in human health assessment
Toxicant
Computational
chemistry,
QSAR
Molecular
Cellular
Initiating event
Responses
Receptor screening assays,
Cell line assays, genomics,
proteomics, metabolomics,
biochemistry
Screening for
Pathway and
toxicological effects and
network impacts
chemicals
Organ
Responses
Whole animal,
In vitro models,
Computational
models, omics,
metabolomics
Mechanistic
modeling
Individual
Responses
Population
Responses
Whole
animal
Toxicology
Population
modeling,
field
monitoring
Predicted
effect
Population
impact
Hypothalamus- Pituitary-Gonadal Axis
Reproductive impairment: Endocrine Disrupting Chemicals
Economic Outcome Pathway
17βtrenbolone
(trb-acetate)
AR binding
AR-dependent
somatic cell
proliferation
Increased muscle
mass
Increased
economic yield
Athletic Outcome Pathway
17βtrenbolone
(trb-acetate)
AR binding
AR-dependent
somatic cell
proliferation
Increased muscle
mass
Improved athletic
performance
Adverse Outcome Pathway: Fathead Minnow
17βtrenbolone
(trb-acetate)
AR binding
testosterone
AR-dependent
somatic cell
proliferation
Tubercle and
fatpad formation
in females
Negative feedback,
hypothalamic
neurons
Reduced
steroidogenesis,
vitellogenesis
estradiol
Reduced
fecundity
vitellogenin
Adverse Outcome Pathway: Human
17βtrenbolone
(trb-acetate)
AR binding
AR-dependent
somatic cell
proliferation
Increased body
hair in females
Negative feedback,
hypothalamic
neurons
Testicular atrophy,
impaired sperm
production
Infertility
Rat versus fathead minnow in vivo tests for endocrine active chemicals
Fathead minnow effective in vivo screening tool
Hypothalamus- Pituitary-Gonadal Axis
Mutual information network based on expression and hormone levels
Androgen
receptor
antagonist
885 microarrays, 9 chemicals, multiple doses, multiple time points
Now over 1700 arrays, more chemicals, more doses
Perkins et al 2011 ET&C
Identification of potential effects on ovaries caused by Flutamide
Physiological changes consistent
with observations in reverse
engineered network
Involvement of SDF-1 in oocyte atresia
-> Potential non-androgenic mediated
effects
Flutamide used as treatment in
polycistic ovary syndrome in
humans ->reduction in ovarian size
and production of androgens
De Leo 1998 JCEM
Understanding simple mixture effects:
Flutamide and trenbolone effects on fathead minnow ovaries
Flutamide
AR antagonist
Mix
Trenbolone
AR agonist
• SDF-1 change in flutamide exposure, but not trenbolone suggests non
androgen-dependent mechanism
• Demonstrates interaction of different androgen and anti-androgenic
chemicals
Toxicity of nitroaromatics in alternative species
Fathead minnow (Pimephales promelas)
Rat (Rattus norvegicus)
Northern Bobwhite quail (Colinus virginianus)
Daphnia magna
Lipids and respiration
2,4DNT
Effect of increasing 2,4 DNT on survival and
liver health
Survival
HI
% Survival
80
8
60
6
40
4
2
20
0
Control Acetone
2
4
8
16
Hepatosomatic Index (HI)
100
Liver lipid metabolism
Liver gene
expression
0
2,4 DNT (mg/L)
Dose and mortality
Wintz et al 2006 Toxicol Sci
Time to Mortality (2,6-DNT Exposure)
350
Time to Mortality (h)
2,6DNT
Liver gene
expression
400
a
a
a
Male
Female
300
250
b
200
c
150
100
50
0
control
50
100
190
350
2,6-DNT Dos e (m g/k g/d)
Dose and mortality
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fatty acid metabolism
glycolysis
gluconeogenesis
anemia
Ruwat et al 2010
Liver lipid metabolism
TNT
24DNT
26DNT
Genomics: Common pathways
impacted by nitrotoluenes
-Nrf2, PPAR alpha, fatty acid
metabolism
-similar to quail and fathead
2ADNT
4ADNT
Liver gene expression
Lipidomics: 37 individual lipid species
affected
Deng et al PLoS ONE 2010
Toxicity of anilines and nitrotoluenes
TypicalO bservations
in R D T Test
Pathw ay
O rgan
/Tisse
Toxicant→
M acro-M olecular Interactions
Hepatocyte
NH2
Liver
1
K upffer Cell:
Phagocytosis
of D am aged
NHOH
Erythrocyte
O xidase
Monooxig
enaze
Nrf2,
PPAR
Erythrocyte
NHOH
2 M et
HG B
Fatty liver
E xtram edullary
hem atopoiesis
Com pensatory
R esponce
Peroxidation
Lipid M em brane
etc.
K upffer Cell:
Hem osiderin
Pigm entation
R B C↓
HG B↓
HT C↓
T. bilirubin↑
3
Erythrocyte
R ed pulp:
Hem osiderin
Pigm entation
E xtram edullary
H em atopoiesis
W hole B ody
E ffects
Ex tram edullary
hem atopoiesis
Hem olysis
4
B one
m arrow
?
O rgan/Tissue
E ffects
R eticulo↑
HG B
A dducts
R ed pulp:
Phagocytosis
of D am aged
Spleen
O rganism
R esponses
M et H G B↑
B lood
NTs
O rgan
Responces
N=O
-H G B
ROS
C ellular
Responses
5
Ex tram edullary
Hem atopoiesis
Congestion
O rgan w t. ↑
Hem atopoiesis
Increased
Hem atopoiesis
Increased
Cyanosis/
Pale Skin
The impact of complex mixtures of nitroaromatics on
Daphnia magna
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RDX
HMX
Aminodinitrotoluenes
Dinitrobenzene
Trinitrobenzene
Trinitrotoluene
Dinitrotoluene
LAAP: Louisiana Army
Ammunition Plant
Garcia-Reyero et al 2011
Effects of individual and mixtures of chemicals on gene expression
Individual chemical effects
DNB
26DNT
Synthetic mixture interactions
TNT
RDX
DNB
TNB
TNT
A26DNT
26DNT
PCR
panel
RDX
26DNT
TNB
Clear mixture effects
D. magna microarray analysis of mixture effects
Pathway analysis (human orthologs)
Mixtures and Individual
chemical pathways
common
pathways
BUT: A number of unique functions were also affected in
mixtures -> consistent with non-additivity of chemical
effects
The top (most connected and significant)
network for each exposure and their
connections
Summary: AOP and alternative animals in human health assessment
Toxicant
Computational
chemistry,
QSAR
Molecular
Cellular
Initiating event
Responses
Receptor screening assays,
Cell line assays, genomics,
proteomics, metabolomics,
biochemistry
Screening for
Pathway and
toxicological effects and
network impacts
chemicals
Organ
Responses
Whole animal,
In vitro models,
Computational
models, omics,
metabolomics
Mechanistic
modeling
Individual
Responses
Population
Responses
Whole
animal
Toxicology
Population
modeling,
field
monitoring
Predicted
effect
Population
impact
• Kurt Gust
• Mitch Wilbanks
• Youping Deng
• Rory Connoly
• Miyuki Breen
University of Louisiana at Monroe
• Sharon Meyer