REACTIVE TOXICITY : PROGRESS REPORT ON FILLING THE GAP Gilman Veith Logan UT March 23-24,2010

REACTIVE TOXICITY:

PROGRESS REPORT ON FILLING THE GAP

Gilman VeithLogan UT

March 23-24,2010

QSAR Foundation Goals

Facilitate promising QSAR technologies for setting priorities

(TIMES-SS, Multipath, ASTER, OECD Toolbox)

Encourage the expansion of public domain databases and software for QSAR applications (ECOTOX, ER mediated toxicity)

Develop high quality databases for QSAR modeling (inhalation for fish and rodents, nucleophile reactivity profiles)

Provide QSAR training for regulators, business experts and students

Logan Workshop Goals Review progress on developing a systematic database for GSH reactivity

Review progress on linking GSH reactivity to important hazard assessment endpoints

Explore progress and options in selecting the next model nucleophile

Explores possibilities for creating next systematic reactivity database.

Purpose of this Overview

Review the context for using QSAR in regulatory safety assessments in relation to drug design

Summarize hazard assessment endpoints which can be modeled by QSAR methods and those that cannot.

Review our conceptual framework for modeling long-term adverse outcomes needed in risk assessment

Summarize progress on integrating QSAR with toxicity pathways for predictive hazard identification.

Initial Hazard Assessments

-Screening Information Datasets – SIDS

-Globally Harmonised System of C&L – GHS

-Registration, Evaluation, Authorisation and Restriction of Chemicals – REACH

-PMNs – OPPT (predictive hazard identification)Testing Requirements – OPP

QSAR ENDPOINTS (SIDS)

Physicochemical Properties and Fate

Melting Point Boiling Point Vapour Pressure Log K o/w Log K orgC/w Water Solubility Biodegradation Rates Hydrolysis Rates Atmospheric Oxidation Rates Bioaccumulation

QSAR ENDPOINTS (~SIDS)Human Health Effects

Acute Oral Toxicity Acute Inhalation Toxicity Acute Dermal Toxicity Skin/Eye Irritation Skin Sensitisation Repeated Dose Toxicity Reproductive Toxicity Developmental Toxicity Genotoxicity (in vitro) Genotoxicity (in vitro, non bacterial) Genotoxicity (in vivo) Carcinogenicity

QSAR ENDPOINTS (SIDS)

Ecological Effects

Acute Lethality – Fish (many species) Chronic Toxicity - Fish Acute Lethality - Daphnid Phytotoxicity, Growth Inhibition -

Algae Repeated Dose Effects - Mammals

QSAR models have been developed for well-defined in vivo endpoints (steady-state exposures)

“Well-defined” excludes most long-term in vivo endpoints and most mammalian tests

>10,000 QSAR models for in vitro endpoints not yet reliably scaled to in vivo potency

QSAR-based Chemical Categories bridge some of these gaps while toxicity pathways are developed

GAPS IN QSAR MODELS

-2 0 2 4 6 8

Log P

-8

-6

-4

-2

0

2Lo

g M

olar

Con

cent

ratio

n

Estimating Aquatic Toxicity

LC50-96hrMATC-30 dayWater Solubility

-2 0 2 4 6 8

Log P

-8

-6

-4

-2

0

2Lo

g M

olar

Con

cent

ratio

n

Estimating Aquatic Toxicity

LC50-96hr

Water Solubility

LogLC50 for fish or rat vs Solubility in water or air

-7

-6

-5

-4

-3

-2

-1

0

-7 -6 -5 -4 -3 -2 -1 0 1 2

Log Solubility, mol/l

Lo

g L

C5

0, m

ol/l

Water Solubility vs FishToxicity

Air Solubility vs RodentToxicity

-2 0 2 4 6 8

Log P

-8

-6

-4

-2

0

2Lo

g M

olar

Con

cent

ratio

n

Framework for Estimating Toxicity

LC50-96hr

Water Solubility

Baseline Toxicity“Excess” Toxicity

Which Conformation should we use to model interactions?

O

CH3

H3C

Energy_LUMO vs PLANARITY_CONJUGATE

-0.2

0

0.2

0.4

0.6

0.8

0 20 40 60 80 100

PLANARITY_CONJUGATE

E_LU

MO

WHY “REACTIVE TOXICITY”?

Nonspecific Narcosis QSAR in 1980 Covers 60-70% of Industrial Chemicals Hundreds of QSARs for Physical Toxicity Largest Gap is Nonspecific Reactive Chemicals Little Progress in Modeling Reactive Toxicity Many Effects Endpoints for of Reactive

Chemicals

MolecularInitiating

Events

Chemical Speciation

and

Metabolism

MeasurableBiological

Effects

Adverse Outcomes

ParentChemical

Our Conceptual Framework

MolecularInitiating

Events

Speciation

and

Metabolism


Effects

Adverse Outcomes

ParentChemical

Our Conceptual Framework

1. Identify Plausible Molecular Initiating Events 2. Design Database for Abiotic Binding Affinity/Rates 3. Develop QSARs to Predict Initiating Event from

Structure 4. Quantify Response Pathways to Downstream

Effects

QSARQSAR ResponseResponsePathways Pathways

Chemistry/Chemistry/BiochemistryBiochemistry

MolecularInitiating

Events

Chemical Speciation

and

Metabolism


Effects

Adverse Outcomes

ParentChemical

Conceptual Framework

Mortality-systemic toxicity

-disease-cancer

Impaired Development

-terata-prenatal deficits

Reproductive Fitness-fertility

-viable offspring

Chemical Inventories andCategories(~200,000)

InteractionMechanisms

-Nonspecific Targets

-Atom CentersTargets

-Receptor Targets

MolecularInitiating

Events

Chemical Speciation

and

Metabolism


Effects

Adverse Outcomes

ParentChemical

At the Molecular Initiating Event

The QSAR Question is:

“How many other chemicalscan interact at this target?”

While the Toxicology Question is:

“What are the known biologicaleffects from this altered target…cell type, organ, species ”

LibraryOf

MolecularInitiating

Events

Chemical Speciation

and

Metabolism


Effects

Adverse Outcomes

ParentChemical

From the Library of Initiating Events

OECD ToolboxOECD ToolboxChemical Chemical

ProfilerProfilerHandles the Handles the

Chemistry for Chemistry for QSAR ModelsQSAR Models

TargetsInteractions

Structural Requirements

Conformations

Metabolic SimulatorsInventories

LibraryOf

MolecularInitiating

Events

Chemical Speciation

and

Metabolism


Effects

Adverse Outcomes

ParentChemical

From the Library of Initiating Events

ERBinding

GeneActivation

ProteinProduction

AlteredGonad

Development

ImpairedReproduction

Mechanistic Profiling

Biological Responses

THE ADVERSE OUTCOME PATHWAY

Toxicant

Molecular Initiating

Event

Chemical Reactivity

Profiles

Chemical Reactivity

Profiles

Receptor, DNA,ProteinInteractions


Macro-Molecular Interactions

Cellular

NRC Toxicological Pathway



Toxicant


Event

Chemical Reactivity

Profiles

Chemical Reactivity

Profiles

Gene Activation

Protein Production

Signal Alteration

Gene Activation

Protein Production

Signal Alteration




Cellular Organ




Toxicant


Event

In Vitro &HTP Screening

Chemical Reactivity

Profiles

Chemical Reactivity

Profiles

Gene Activation

Protein Production

Signal Alteration

Gene Activation

Protein Production

Signal Alteration

AlteredFunction

Altered Development

AlteredFunction

Altered Development




Cellular Organ


In VivoTesting



Toxicant Organism


Event

In Vitro &HTP Screening

Chemical Reactivity

Profiles

Chemical Reactivity

Profiles

Gene Activation

Protein Production

Signal Alteration

Gene Activation

Protein Production

Signal Alteration

AlteredFunction

Altered Development

AlteredFunction

Altered Development

Lethality

Sensitization

Birth Defect

Reproductive Impairment

Cancer

Lethality

Sensitization

Birth Defect

Reproductive Impairment

Cancer

Structure

Extinction

Structure

Extinction



Macro-Molecular Interactions Population

MAJOR PATHWAYS FOR REACTIVE TOXICITY FROM MODERATE ELECTROPHILES

Systemic Responses

SkinLiverLung

Systemic Responses

SkinLiverLung

MichaelAddition

Schiff baseFormation

SN2

Acylation

MichaelAddition


SN2

Acylation

AtomCentered

Irreversible(Covalent)Binding

AtomCentered



MolecularInitiatingEvents Exposed

SurfaceIrritation

ExposedSurface

Irritation

SystemicImmune

Responses

SystemicImmune

Responses

Necrosis:Which

Tissues?

In vivoEndpoints

Pr-S AdductsGSH OxidationGSH DepletionNH2 AdductsRN AdductsDNA Adducts


Oxidative

Stress

Oxidative

Stress

Dose-Dependent Effects

R 394

E 353 H 524

BBAA

Representation ofRepresentation of ER binding ER binding pocket (LBD), with 3 sites of pocket (LBD), with 3 sites of interaction shown (A, B, C), interaction shown (A, B, C), and recepter protein amino and recepter protein amino acids involved in interactions acids involved in interactions with chemical ligands.with chemical ligands. CC

T 347

CC

J. Katzenellenbogen

R 394

E 353 H 524

CC

T 347

HOOH

CH3 H

H H

H

AA BB

A_B Interaction A_B Interaction

Distance = 10.8 for 17-EstradiolJ. Katzenellenbogen

ReproductiveImpairment

Adverse Outcome PathwayER-mediated Reproductive ImpairmentMeasurements across levels of biological organization

In vivo

INDIVIDUAL

Sex reversal;

Altered behavior;

Repro.


In vivo

INDIVIDUAL

Skewed Sex

Ratios;

Yr Class

POPULATION

Liver Altered gene products

(timing, amt)

Gonad Ova-testis; Sex-reversed; Fecundity

Sex reversal;

Altered behavior;

Repro.


In vivo

TISSUE/ORGAN INDIVIDUAL

Skewed Sex

Ratios;

Yr Class

POPULATION

Liver CellsAltered Protein

Expression(marker)(effect)

Vitellogenin


(timing, amt)


Sex reversal;

Altered behavior;

Repro.


In vivo

CELLULARResponse


Skewed Sex

Ratios;

Yr Class

POPULATION

ReceptorBinding

ER-Chemical Binding

Liver CellsAltered Protein

Expression(marker)(effect)

Vitellogenin


(timing, amt)


Sex reversal;

Altered behavior;

Repro.


In vivo

MOLECULAR Target

CELLULARResponse


Skewed Sex

Ratios;

Yr Class

POPULATION

QSAR focus area

Chemicals

ReceptorBinding

ER Binding

Liver CellsAlteredProtein

Expression

Vitellogenin

LiverAltered proteins

GonadOva-testis;

Sex-reversed;Fecundity

Sex reversal;

Altered behavior;

Repro.

Adverse Outcome PathwayER-mediated Reproductive Impairment

Measurements made across levels of biological organization

In vivo

MOLECULAR Target

CELLULARResponse


Skewed Sex

Ratios;

Yr Class

POPULATION

In vitro Assayfocus area

Risk Assessment RelevanceToxicological Understanding

35

ER-mediated Adverse-outcome PathwayAmylaniline (AAN)

Molecular Cellular Organ Individual Population• AAN binding

to ER• Liver slice

Vtg (mRNA)

• Liver slice toxicity

•Altered reproduction

•Altered developmentDecreased numbers of animals

In-vitro pathwaySchmieder et.al.

• ER transcription factor

In-vivo pathwayMultigen assay

dose: Sex reversal (altered gamete ratios)

Population reduction

AAN bindingto ER

ER transcriptionfactors

♂ Liver Vtg (mRNA)

Anal fin papillae?

Gonadal morphology??

dose: Mixed-sex gonad

Molecular PopulationCellular IndividualOrgan

Altered sex-ratios?

AAN bindingto Hbg ?

Splenic/head-kidney pathology

?

dose: Reduced fecundity

dose: Reduced growth ?

?

ThyroperoxidaseIodine Symporter

Exposure

Hepatic UDPGTs

Thyroidal

Extra-Thyroidal

Deiodinases

Thyroid Receptors

T4–TTR Binding

Targets

Cellular Transporters

EarlyBiological

Effect

TissueSpecificEffect

Altered Structure/Function

ClinicalDisease

SerumT3 & T4

Changes

TSH

TissueT3 Changes

AlteredDevelopment

ThyroidHyperplasia

ThyroidTumors

BirthDefects

EffectsCancer & Non-Cancer

Thyroid MOAs

HEARING LOSS FROM DIOXINS, FURANS AND PCBS (PLANAR RISK)

Exposure Hepatic Phase II Enzymes

Hepatic Parent or Metabolite

SerumT4 & T3

TissueT3

Alter TR Mediated Proteins

Loss of cochlear hair cells

HearingLoss

Binding to PXR

Binding to AhR

“Narcosis” Pathways for Volatile Anesthetics

nACh

Primary Brain

Region

Behavioral Effects

Hippocampus

Light SedationAmnesia

Anxiolysis

Ion Channel Receptor

Agent

CellularResponse

GABAA

Glycine

NMDA

Decreased channel-open time

Reduced membrane current

Increased channel-open time

Increased duration of mIPSCs

TissueResponse

Reduced excitatory transmission


Facilitated inhibitory transmission


Cortex - Thalamus

Brain Stem

Spinal Cord

Unconsciousness Loss of perceptual

awareness

Heavy Sedation Slow responses

Immobility Loss of pain

response

Increasin

g Depth

of An

esthesia

Kinetics Dynamics

nACh

Immobility Pathway for IsofluranePrimary

Brain Region

Behavioral Effects

Hippocampus

Light SedationAmnesia

Anxiolysis


Agent

CellularResponse

GABAA

Glycine

NMDA





TissueResponse





Cortex - Thalamus

Brain Stem

Spinal Cord


awareness



response

Increasin

g Depth

of An

esthesia

Kinetics Dynamics

nACh

Amnesia Pathway for Isoflurane Primary

Brain Region

Behavioral Effects

Hippocampus

Light SedationAmnesia Anxiolysis


Agent

CellularResponse

GABAA

Glycine

NMDA





TissueResponse





Cortex - Thalamus

Brain Stem

Spinal Cord


awareness



response

Kinetics Dynamics

Increasin

g Depth

of An

esthesia

Effectopedia

Cause Link Effect

Pathways for Reactive Toxicity

MichaelAddition


SN2

Acylation

AtomCentered



MolecularInitiatingEvents

Membrane Alteration

___

Oxidative Stress

___

Genotoxicity

Death

ImpairedGrowth

Impaired Development

Impaired Reproduction

In vivoEndpoints


Dose-Dependent PathwaysSpecies/Sex/Life-Stage

In vitroEndpoints

Two Questions for Building Pathways

Pr-S Adducts

GSH Oxidation

GSH Depletion

NH2 Adducts

RN Adducts

DNA Adducts

Effect#1

Effect#2

Effect#3

Direct Reaction

AlteredSynthesis

Oxidation

How Many Ways to Deplete GSH? How Many Downstream Effects?

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

Chemical Reactivity

Profiles

Reversible Nonspecific

Binding

ReversibleSpecific Binding

CovalentBinding

Lethality

Growth

Development

Reproduction

Molecular/Subcellular Cell Organ Individual

In Silico Methods In vitro Methods In vivo Methods

Electronic

MolecularInitiating

Events

Membranes

EnergyCharge

NuclearReceptors

ProteinSynthesis

DNAIntegrity

ChemicalInventories

Response Pathways RegulatoryEndpoints

Exposure/Metabolism

PenetrationRoutes

DetoxificationPathways

ActivationPathways

More Relevant Endpoints

Better Defined Endpoints

Intrinsic ChemicalAttributes

Tissue

Major Pathway for Reactive Toxicants To Fish

MichaelAddition


SN2

Acylation

Atom Centered Irreversible(Covalent)

ProteinBinding


MolecularInitiatingEvents

“AnyExposedSurface”Changes

Necrosis of the Gill Epithelium

In vivoEndpoints

Pathogenesis

Vulnerable Organ Patholog

y

Death from

Suffocation

ComplexesMembranes,

etc

Pathways for Reactive Toxicity from Soft Electrophiles

Systemic Responses

SkinLiverLung

MichaelAddition


SN2

Acylation

AtomCentered

Irreversible(Covalent)

ProteinBinding

Immunogenic

MechanismsMolecularInitiatingEvents

ExposedSurfaceIrritation

SystemicImmune

Responses

NecrosisSkin

Lung/GillsGI Tract

In vivoEndpoints

Yes

No

Major Pathways for Reactive Toxicity from Moderate Electrophiles

Systemic Responses

SkinLiverLung

MichaelAddition


SN2

Acylation

AtomCentered



MolecularInitiatingEvents Exposed

SurfaceIrritation

SystemicImmune

Responses

NecrosisWhich

Tissues?

In vivoEndpoints


Oxidative Stress

Dose-Dependent Effects

Simulated 2-Acetylaminofluorene

Metabolism

NH

O

NH

O

OH

NH

O

O

NH2

O

HO

O

NHOH

O

N+HO

NH

OHO

NH

O

O

NH

O

O

NH

OHO

NH

OHO

OHNH

OHO

OH

NH

OHO

O

NH

OHO

O

N+H

HO

ON+H

OH

O

. . . . . .

NHX

OO

X = H, OH,

O

Activated metabolites

Which Metabolite should we use in modeling interactions?Which Metabolite should we use in modeling interactions?

Fish and mammal inhalation baseline toxicity are not directly comparable because the external media are different

However, blood thermodynamic activity for LC50(nar) should be the same in fish and mammal

At steady-state, the activity in air/water equals the activity in blood by definition :

α = С x γα – activity; C- concentration; γ-activity

coefficient

Baseline Toxicity

The thermodynamic activity at any concentration can be estimated by dividing by the solubility in the medium

activity for narcosis in fish = LC50(fish)/water solubility

activity for narcosis in rat = LC50 (rat)/air solubility

if activity for narcosis in fish and rat were equal, the plot of LC50 versus solubility in exposure medium should be the same

Baseline Toxicity

Quenching

Overload?

ChemicalInventories

NonreactiveFamilies

Detoxication

QSARLibrary (24,000)

Excretion

ReactiveFamilies

Structures

Low

Chemical PropertiesNon-Specific Pathways

Receptor-Based Pathways

DNADamage

AntigenicConjugate

CriticalCellularTargets

High

Immune Response

Mutagenicity

Carcinogenicity

Necrosis

Deterministic Endpoints

Probabilistic Endpoints

Genome-Specific Endogenous Factors

Test Method-Specific Factors

ConformationalAnalysis

Virtual Metabolism

?

?

PROBABILISTIC MODELS

Peffect = P1 x P2 x P3 x P4 x …Pn

Exposure of the individual Delivery rate to liver Formation of reactive metabolites Exceed detoxification rates Covalent binding with proteins

Formation of neoantigens Immune system recognition Formation of cytotoxic antibodies Interaction with hepatocytes Overwhelm repair mechanisms

Liver Function Impairment Liver Failure

--after Li (2002)

Forecasting distinct probabilities of low incident outcomes like idiosyncratic hepatic failure requires probability distributions for critical steps rather than effects under standard conditions

PROBABILISTIC MODELS FOR PRIORITIZATION

Peffect = Pchem x Pexposure x Penviron x Pgenetic

Risk Management

Scenarios

Chemical Reactivity

Profiles

Prioritization does not require explicit estimates of toxicity but rather a reliable ordering with respect to explicit risk management scenarios

Documents

REACTIVE TOXICITY : PROGRESS REPORT ON FILLING THE GAP Gilman Veith Logan UT March 23-24,2010