The Adverse Outcome Pathway Approach toEcotoxicology and Its Possible Applicability to

Human Health Toxicology

Ed Perkins, PhDSenior Research Scientist Environmental Laboratory

US Army Engineer Research and Development Center

US Army Corps Engineers

Vicksburg, MS

email: edward.j.perkins@usace.army.mil

Mixtures and Cumulative Risk Assessment: New Approaches: Using the Latest Science and Thinking about Pathways

July 27–28, 2011

Raw Material

Lifetime Article

Env. Fate

Eco health

Human health

Alt species

Pathway modeling Rapid in vivo screeningSystems toxicology and molecular analysis

• 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

Application of Research to Levels of Organization Based

on Source to Outcome Source

EnvironmentalContaminant

Exposure

Key Event Cellular Effects

Individual

Population

Community

Mode of Action

Adverse Outcome Pathway

Source to Outcome Pathway

Toxicity Pathway

4

Biologicinputs

“Normal” BiologicalFunction

AdverseOutcomes

(e.g., mortality, ReproductiveImpairment)

Cell inury, Inability to

regulate

AdaptiveResponses

Early cellularchanges

Exposure

Uptake-Delivery to Target Tissues

Perturbation

Cellular response pathway

Molecularinitiating event

Perturbed cellular response pathway

Adverse outcomerelevant to

risk assessment

Toxicity Pathway

Adverse Outcome Pathway

An Adverse Outcome Pathway (AOP) is a conceptual framework that portrays existing An Adverse Outcome Pathway (AOP) is a conceptual framework that portrays existing

knowledge concerning the linkage between a direct knowledge concerning the linkage between a direct molecular initiating eventmolecular initiating event and an and an

adverse outcomeadverse outcome, at a level of biological organization relevant to risk assessment., at a level of biological organization relevant to risk assessment.

((AnkleyAnkley et al. 2010, Environ. et al. 2010, Environ. ToxicolToxicol. Chem., 29(3): 730. Chem., 29(3): 730--741.)741.)

ChemicalProperties

Receptor/LigandInteraction

DNA Binding

Protein Oxidation

Gene activation

Protein production

Altered signaling

Altered physiology

Disrupted homeostasis

Altered tissue development/

function

Lethality

Impaired Development

Impaired Reproduction

ToxicantCellular

ResponsesOrgan

ResponsesOrganismResponses

Structure

Recruitment

Extinction

PopulationResponses

Macro-Molecular

Interactions

Toxicant MolecularInitiating event

Cellular Responses

OrganResponses

IndividualResponses

PopulationResponses

Computational chemistry,

QSAR

Receptor screening assays, Cell line assays, genomics, proteomics, metabolomics, biochemistry

Whole animal, In vitro models,Computational models, omics, metabolomics

Whole animal Toxicology

Population modeling,field monitoring

Mechanistic modeling

Predicted effect

Population impact

Pathway and network impacts

Screening for toxicological effects and

chemicals

AOP and alternative animals in human health assessment

Hypothalamus- Pituitary-Gonadal Axis

17β-trenbolone

(trb-acetate)AR binding

AR-dependent somatic cell proliferation

Increased muscle mass

Increased economic yield

Economic Outcome Pathway

Athletic Outcome Pathway

17β-trenbolone

(trb-acetate)AR binding

AR-dependent somatic cell proliferation

Increased muscle mass

Improved athletic performance

Reproductive impairment: Endocrine Disrupting Chemicals

Adverse Outcome Pathway: Fathead Minnow

17β-trenbolone

(trb-acetate)AR binding

Negative feedback, hypothalamic

neurons

AR-dependent somatic cell proliferation

Tubercle and fatpad formation

in females

Reduced steroidogenesis,

vitellogenesis

Reduced fecundity

Adverse Outcome Pathway: Human

17β-trenbolone

(trb-acetate)AR binding

Negative feedback, hypothalamic

neurons

AR-dependent somatic cell proliferation

Increased body hair in females

Testicular atrophy, impaired sperm

productionInfertility

testosterone estradiol vitellogenin

Rat versus fathead minnow in vivo tests for endocrine active chemicals

Fathead minnow effective in vivo screening tool

Hypothalamus- Pituitary-Gonadal Axis

885 microarrays, 9 chemicals, multiple doses, multiple time points

Now over 1700 arrays, more chemicals, more doses Perkins et al 2011 ET&C

Mutual information network based on expression and hormone levels

Androgen receptor antagonist

Physiological changes consistent with observations in reverse engineered network

Involvement of SDF-1 in oocyte atresia-> Potential non-androgenic mediated effects

Identification of potential effects on ovaries caused by Flutamide

Flutamide used as treatment in polycistic ovary syndrome in humans ->reduction in ovarian size and production of androgens

De Leo 1998 JCEM

Flutamide and trenbolone effects on fathead minnow ovaries

FlutamideAR antagonist

TrenboloneAR agonist

Mix

• SDF-1 change in flutamide exposure, but not trenbolone suggests non androgen-dependent mechanism

• Demonstrates interaction of different androgen and anti-androgenic chemicals

Understanding simple mixture effects:

Toxicity of nitroaromatics in alternative species

Rat (Rattus norvegicus)

Northern Bobwhite quail (Colinus virginianus)

Fathead minnow (Pimephales promelas)

Daphnia magna

Wintz et al 2006 Toxicol Sci

2,4DNT

Hepatosom

aticIndex (H

I)

8

6

4

2

0

Effect of increasing 2,4 DNT on survival and liver health

0

20

40

60

80

100

Control Acetone 2 4 8 162,4 DNT (mg/L)

% S

urvi

val

SurvivalHI

Liver gene expression

Liver lipid metabolism

Lipids and respiration

2,6DNTTime to Mortality (2,6-DNT Exposure)

0

50

100

150

200

250

300

350

400

control 50 100 190 350

2,6-DNT Dose (m g/kg/d)

Tim

e to

Mor

talit

y (h

)

Male

Female

a a a

bc

Dose and mortality

Dose and mortality

Liver gene expression • fatty acid metabolism

• glycolysis• gluconeogenesis • anemia

Ruwat et al 2010

TNT24DNT26DNT

2ADNT4ADNT

Genomics: Common pathways impacted by nitrotoluenes-Nrf2, PPAR alpha, fatty acid metabolism -similar to quail and fathead

Liver gene expression

Liver lipid metabolism

Lipidomics: 37 individual lipid species affected

Deng et al PLoS ONE 2010

Organ/Tisse

Toxicant→M acro-M olecular Interactions

CellularResponses

OrganResponces

OrganismResponses

Organ/TissueEffects

W hole BodyEffects

Liver

Blood

Spleen

Bonem arrow

Pathw ayTypical Observations

in RDT Test

Hepatocyte

Erythrocyte

R ed pulp:

Phagocytosisof Dam aged

Erythrocyte

Extram edullary

hem atopoiesis

Hem atopoiesis

Increased

Extram edullary

Hem atopoiesis

T. bilirubin↑

R BC↓

HG B↓HTC↓

R eticulo↑

M et HG B↑

Cyanosis/

Pale Skin

K upffer Cell:

Hem osiderinPigm entation

Extram edullary

hem atopoiesis

R ed pulp:

Hem osiderinPigm entation

Extram edullary

Hem atopoiesisCongestion

O rgan w t. ↑

Hem atopoiesis

Increased

K upffer Cell:

Phagocytosisof Dam aged

Erythrocyte

Com pensatory

R esponce

1

2

3

45

Hem olysis

NHOHHG B

M et -HGB

N=O

ROS

HG BA dducts

PeroxidationLipid M em braneetc.

NHOHNH2

OxidaseMonooxigenaze

Toxicity of anilines and nitrotoluenes

Fatty liver

Nrf2, PPAR

?

NTs

The impact of complex mixtures of nitroaromatics on Daphnia magna

LAAP: Louisiana ArmyAmmunition Plant

• RDX• HMX• Aminodinitrotoluenes • Dinitrobenzene• Trinitrobenzene• Trinitrotoluene • Dinitrotoluene

Garcia-Reyero et al 2011

Individual chemical effects Synthetic mixture interactions

RDX DNB TNB TNT A26DNT 26DNT

RDX

TNT

TNB

DNB

26DNT

26DNT

Effects of individual and mixtures of chemicals on gene expression

Clear mixture effects

PCR panel

The top (most connected and significant) network for each exposure and their connections

Mixtures and Individual chemical pathways

BUT: A number of unique functions were also affected in mixtures -> consistent with non-additivity of chemical

effects

common pathways

Pathway analysis (human orthologs)

D. magna microarray analysis of mixture effects

Toxicant MolecularInitiating event

Cellular Responses

OrganResponses

IndividualResponses

PopulationResponses

Computational chemistry,

QSAR

Receptor screening assays, Cell line assays, genomics, proteomics, metabolomics, biochemistry

Whole animal, In vitro models,Computational models, omics, metabolomics

Whole animal Toxicology

Population modeling,field monitoring

Mechanistic modeling

Predicted effect

Population impact

Pathway and network impacts

Screening for toxicological effects and

chemicals

Summary: AOP and alternative animals in human health assessment

• Kurt Gust• Mitch Wilbanks• Youping Deng

University of Louisiana at Monroe• Sharon Meyer

• Rory Connoly• Miyuki Breen