08 P Schmieder Toxicity Pathways to Assessment Endpointsmckim.qsari.org/Presentations/08_P_Schmieder_Toxicity Pathways to... · Toxicity Pathways to Assessment Endpoints P. Schmieder,

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Toxicity Pathways to Assessment Endpoints

P. Schmieder, S. Bradbury, G. Veith, J. McKim

Toxicity Pathway

• A concept; a way of depicting a chain of events starting with a molecular initiating event (site of chemical –biological interaction) and ending with an adverse effect manifested in an individual, or higher level – population, community, ecosystem

• May include a biochemical/signaling pathway, but goes beyond, to at least hypothesize how something observed at one level of biological organization is linked to response manifested at another level.

• Chemical similarity is defined in the context of biological similarity

– “Similar” chemicals, by definition, invoke the same toxicity pathway (within a specified biological model)

– QSARs are developed for “similar” chemicals from a known or hypothesized “mode/mechanism” of action; hypothesis is tested torefine the models

• QSAR requires a well-defined biological system

WHAT:

WHY:

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Effects of toxicants occur at different levels of biological

organization. Toxic effects are best known and understood at

the cell and organ level, while the ecosystem and community

level are least understood although most relevant.

(Haux and Forlin, 1988)

Productivity

Energy Flow

Population CellOrganIndividual

Understanding

Relevance

CommunityEcosystem

Contaminant

dynamics in

microcosms

Chronic

toxicity

Reproduction

Growth

Acute toxicity

Lethal

Sublethal

Respiration

Osmoregulation

Structural

changes

Induction

TOXIC

CHEMICAL

Toxicity Pathway Uses

• Assess knowledge gaps - what we know and what we don’t know about a chemical’s toxicity (toxicodynamics)

• Assess the plausibility that a series of events are linked, i.e., degree of connectedness; – degree of specificity/certainty needed depends upon intended use

• prioritization for further testing – correlation; “good” hypothesis?• quantitative RA - confirm cause and effect?

• Pinpoint molecular initiating event for chemical extrapolation– QSAR – can be based on in vivo endpt if system is simple enough,

e.g., fish acute/chronic for narcotic chemicals where applied chem concis directly related to chemical activity in blood and further to the whole organism effect

– Measurements closer to molecular initiating event will be more definitive for QSAR but some degree of relevance should be established (Linkage across levels of biological organization)

• Basis for species extrapolation

• Shifting RA paradigm - predict most likely tox pathways for a chemical to pinpoint most appropriate testing

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Well-Defined Biological System(Know what you know and what you don’t know)

• Metabolism– Is the system used for collection of empirical data

capable of xenobiotic metabolism?

– Is what you’re measuring due to parent chemical or a metabolite?

• Kinetics– What do you understand about the chemical kinetics

within the system?

– Is the chemical in solution• Bound and unavailable

• Loss to hydrolysis

Measure chemical form and concentration in your system

-2 0 2 4 6-8

-6

-4

-2

0

Log P

Lo

g F

ah

ea

d M

inn

ow

Mo

lar

To

xic

ity (

1/L

C5

0)

4

-2 0 2 4 6 8

Log P

-10

-8

-6

-4

-2

0

Lo

g F

ath

ea

d M

ola

r T

ox

icit

y (

1L

C5

0)

Fathead Minnow Acute Toxicity Database

Narcosis I

Narcosis IINarcosis III

Uncoupler

Sorting Modes of Action

(Toxicity Pathways)

Fish Acute Toxicity Syndromes

- respiratory/cardiovascular responses (RBT)

Behavioral observations (FHM)

Mixture studies (FHM)

5

Nonpolar Narcotic Toxicants

-7

-6

-5

-4

-3

-2

-1

0

0 1 2 3 4 5 6

Log P

Lo

g M

ola

r C

on

ce

ntr

ati

on

LC50-96hr MATC-30 day Water Solubility

Assigning ChemToxicol. Similarity

for QSAR

Xenobiotic

MembranePartitioning

Ion Gradient

Interruption

Failed ATP

Production

-Decreased Respiration

-Decreased Circulation

-Faulty Osmoregulation

Delineating Toxicity Pathways Across Levels of Biological Organization:

Acute Nonpolar Narcosis

Toxicological

Understanding

Risk Assessment

Relevance

In vivo Assays

MOLECULARTARGETS/RESPONSES

TISSUE/ORGAN SYSTEM PHYSIOLOGYINDIVIDUAL

Lethality

6

-2 0 2 4 6 8

Log P

-8

-6

-4

-2

0

2

Log M

ola

r C

oncentr

ation

Water SolubilityLC50-96hrMATC-30 day

LC50-96hr

MATC-30 day

Uncoupling Toxicants


for QSAR

Xenobiotic

Chemical

Partitioning

Membrane

Proteins/

Ion Channels

-Increased Respiration

-Increased O2 Consumption

-Decreased O2 Utilization


Acute Uncoupling of Oxidative Phosphorylation

Toxicological

Understanding

Risk Assessment

Relevance

In vivo Assays

MOLECULARTARGETS

TISSUE/ORGAN SYSTEM PHYSIOLOGYINDIVIDUAL

Lethality

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Reactive Toxicants

-8

-7

-6

-5

-4

-3

-2

-1

0

0 1 2 3 4 5 6

Log P

Lo

g M

ola

r C

on

ce

ntr

ati

on

LC50-96hr MATC-30 day Water Solubility

Sorting Modes of Action

(Toxicity Pathways)

Fish Acute Toxicity Syndromes

- respiratory/cardiovascular responses (RBT)

Behavioral observations (FHM)

Mixture studies (FHM)

Biochemical responses – in vitro

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Effects of toxicants occur at different levels of biological

organization. Toxic effects are best known and understood at

the cell and organ level, while the ecosystem and community

level are least understood although most relevant.

(Haux and Forlin, 1988)

Productivity

Energy Flow

Population CellOrganIndividual

Understanding

Relevance

CommunityEcosystem

Contaminant

dynamics in

microcosms

Chronic

toxicity

Reproduction

Growth

Acute toxicity

Lethal

Sublethal

Respiration

Osmoregulation

Structural

changes

Induction

TOXIC

CHEMICAL


for QSAR

Xenobiotic

Binding to cytoskeletalcomponents

-Redox cycling- SH Arylation

GSH OxidationPrSH OxidationROS Production

Decr. Energy ChgDisrupt Cytoskel.

(MT;IF);Blebbing

Altered Cell SignalingCell Death

Liver Toxicity

Multiple Organ

System

Toxicities/Disease

Defining Toxicity Pathways Across Levels of Biological Organization:

Redox cycling_Arylation

Toxicological

Understanding

Risk Assessment

Relevance

In vivo AssaysIn vitro Assays

MOLECULAR

CELLULAR

TISSUE/ORGANINDIVIDUAL

Lethality

Impaired

Growth

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Non-halogenated alkanes

Halogenated alkane/enes

Ethers

Alcohols

Aldehydes

Ketones

Esters

Nitriles

Aliphatic amines

Aromatic amines

Azides

Sulfides/thiols

Benzenes

Phenols

Nitro-ring

5-Membered ring

Hetero atom

Sulfur compound

Anilides/ureas

Phosphates

Pesticides

Pharmaceuticals

0 10 20 30 40 50 60

6

25

23

55

39

31

47

15

6

31

2

12

21

37

28

9

6

1

5

4

28

9

Chemical Class

Number of Chemicals

Narcosis I

Narcosis II

Narcosis III

Oxidative Phosphorylation Uncouplers

AChE Inhibition

Respiratory Inhibition

Electrophile/proelectrophile Reactivity Mechanisms

CNS Seizure Mechanisms

Chemical Class is not MOA for Industrial Chemical Acute Tox

Molecular

Initiating Events

Speciation

and

Metabolism

Measurable

System Effects

Adverse Outcomes

ParentChemical

Knoxville Workshop Framework for Predicting Reactive Toxicity

Rather than developing statistical models of complex endpoints,

molecular initiating events are identified as well-defined QSAR

endpoints…..and used to estimate the probabilities for important

downstream biological effects based on transparent

assumptions

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Molecular

Initiating Events

Speciation

and

Metabolism

Measurable

System Effects

Adverse Outcomes

ParentChemical

Steps to the Development of QSAR for Steps to the Development of QSAR for

Reactive ToxicantsReactive Toxicants

1. Establish Plausible Molecular Initiating Events2. Design Database for Abiotic Binding Affinity/Rates

3. Explore Correlations/Pathways to Downstream Effects4. Explore QSARs to Predict Initiating Event from Structure

QSARQSAR Systems Systems

BiologyBiology

Delineation of Toxicity PathwaysDelineation of Toxicity PathwaysLinkages Across Levels of Biological OrganizationLinkages Across Levels of Biological Organization

Chemical Reactivity

Profiles

Receptor binding

DNA alterationProteins adducts

Membrane effects

Gene Activation

Protein Syn/deg Cell Signaling

GSH balance

Respiration

OsmoregulationLiver Function

Gonad Devel

Lethality

GrowthDevelopment

Reproduction

Molecular Cellular Organ Individual

In Silico MethodsIn vitro Methods In vivo Methods

Electronic

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Endocrine Disruptors:-Receptor-Mediated Toxicity Pathways

ER, AR, TR?-Enzyme Inhibition (aromatase)-Steroidogenesis (altered steroid metab)

Understanding “Specific” Toxicities

QSAR

Xenobiotic

ER Binding

Altered

ProteinExpression

Altered Hormone Levels,

Ova-testis

Chg 2ndry Sex Char,

Altered

Repro.


Direct Chemical Binding to ER

Toxicological

Understanding

Risk Assessment

Relevance

In vivo AssaysIn vitro Assays

MOLECULARCELLULAR

TISSUE/ORGANINDIVIDUAL

Skewed

Sex Ratios,AlteredRepro.

POPULATION

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Peripheral

TissuesDeiodination

Morphology

HypothalamusTRH (CRH) Release

Thyroid GlandThyroid Hormone

Synthesis

Pituitary

GlandTSH Release

Xenopus Metamorphosis Model for Thyroid System Disruption

Gene/Protein

ExpressionCirculating TH Status Thyroid Histology Altered Morphology

Molecular

Cellular Tissue Individual

Conceptual Overview of Project

Increasing Ecological

Relevance

Increasing Diagnostic

(Screening) Utility

Phase 1.

Fathead minnow 21 d reproduction test

Phase 2.

Zebrafish

genomics

proteomics

Population

modeling

Phase 3.

Fathead minnow

molecular markers

metabonomics

HPG Systems modeling

Molecular•Gene expression

•Protein levels•Receptor binding

•Enzyme activities

CellularAlterations in

production of signalling molecules

Organ•Functional changes

•Structural changes

(Pathology)

IndividualAltered reproduction

or development

PopulationDecreased numbers

of animals

Levels of

Biological

Organization

Small teleost model, well characterized

genome, low ecological /

regulatory relevance

Computational

modeling

Small teleost model, poorly characterized

genome, high ecological /

regulatory relevance

→’s Depict the flow of information

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Chemical Risk Assessments

Chemical 2-D

Structure/Properties

Chemical 3-D


Receptor/

LigandInteraction

Gene

Activation

Protein Production

Gonad

Development

(Ova-Testis)

Altered Hormone

Levels

Impaired Kidney

Function

Impaired

Reproduction

Linkages Across Levels of Biological Organization

Molecular Cellular

Organ

Individual

Understanding

Relevance

Receptor-Mediated Pathways

Metabolism

In vivo

ToxicokineticsToxicodynamics

Chemical-Receptor BindingInitiatingAltered

Gene/ProteinExpression

Changes in Gene/ProteinExpression

Leading to Altered Cell

Function

Altered Organ

Growth and Function

ImpairedReproduction

Cell Organ/Tissue IndividualXenobioticChemical

Molecular/

Sub-Cellular

Toxicological

Understanding

Risk Assessment

Relevance

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In vivo

Chemical Kinetics

Receptor Binding

Gene/Protein

Expression

Gene/Protein

Cell Function

Growth and

FunctionReproduction

Cell Organ/Tissue IndividualXenobioticChemical

Molecular/

Sub-Cellular

Toxicological

Understanding

Risk Assessment

Relevance

In vivo

Chemical Kinetics

Trout

Cell Organ/Tissue Individual

XenobioticChemical

Molecular/

Sub-Cellular

Toxicological

Understanding

Risk Assessment

Relevance

Uptake

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In vivo

Chemical Kinetics

Trout


XenobioticChemical

Molecular/

Sub-Cellular

Toxicological

Understanding

Risk Assessment

Relevance

UptakeDistribution/Metabolism

In vivo

Chemical Kinetics

Trout


XenobioticChemical

Molecular/

Sub-Cellular

Toxicological

Understanding

Risk Assessment

Relevance

UptakeDistribution/Metabolism/Excretion

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In vivo

Metabolism studies across levels of biological organizationLinkages must be established

MicrosomesS9

Purified enzymes

IsolatedHepatocytes

Celllines

Isolated Perfused

Liver

Tissue Slices

Trout


XenobioticChemical

Molecular/

Sub-Cellular

Toxicological

Understanding

Risk Assessment

Relevance


In vitro

Time (h)

0 4 8 12 16 20 24 28

Pe

rcen

tag

e o

f M

ax

imu

m 3

H-E

str

ad

iol

0

20

40

60

80

100

120

Slices: Ethylacetate Fraction

Slices: Aqueous Fraction

RTH149 Cells: Ethylacetate Fraction

RTH149 Cells: Aqueous Fraction

Metabolism of EstradiolRainbow Trout Liver Slices vs RTH149 Cells

(E2)

(E2-gluc)

(E2)

(gluc)

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In vivo

Chemical Kinetics

Receptor Binding

Gene/Protein

Expression

Gene/ProteinExpression

Cell Function

Growth and

FunctionReproduction


XenobioticChemical

Molecular/

Sub-Cellular

Toxicological

Understanding

Risk Assessment

Relevance


OPP ChemicalsPredicted inactive

parent;

“activated”

metabolites

Predicted Metabolites

Existing Metabolism

Simulator

Existing ER

Binding Model

Prioritized Chemicals

Verified maps

Project Goal: Enhance Metabolic Simulator for EPA Regulatory Lists

Trout

liver slice

Rat liver microsomes,S9

enhance

Expert Judgement

Analytical

methods

Verified ER

activation

Existing E

R

Binding Model

simulator

improveER model

MED; NERL-Athens; LMC

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Toxicity Pathways

A useful concept for organizing toxicity data across levels of

biological organization

-Linking toxicological understanding to risk assessment relevance

A conceptual framework for:

- chemical extrapolation

- molecular initiating events are the key to linking chemical reactivity continuum to biological

response continuum

- species extrapolation

A useful concept in Predictive Toxicology

- Predict most likely tox pathway for a chemical to

pinpoint most appropriate testing

ER Binding ER TransctivationVTG mRNA

Vitellogenin InductionSex Steroids

AlteredReproduction/DevelopmentMolecular Cellular Organ Individual

Chemical 3-DStructure/Properties

Chemical 2-D StructureStructure







































































Libraries of Toxicological Pathways

ER Binding

ER Transctivation

VTG mRNA

VitellogeninInduction

Sex Steroids

AlteredReproduction/Development

Molecular Cellular Organ Individual

Chemical 3-D


Chemical 2-D

Structure

Structure

Init

iati

ng

Eve

nts

Imp

air

ed

Re

pro

du

cti

on

/De

velo

pm

en

t

Mapping Toxicity Pathways to Adverse Outcomes

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Libraries of Toxicological Pathways

Init

iati

ng

Eve

nts

Ad

ve

rse

Ou

tco

mes

Mapping Toxicity Pathways to Adverse

Outcomes

Documents

08 P Schmieder Toxicity Pathways to Assessment Endpointsmckim.qsari.org/Presentations/08_P_Schmieder_Toxicity Pathways to... · Toxicity Pathways to Assessment Endpoints P. Schmieder,