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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
MeasurableBiological
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
MeasurableBiological
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
MeasurableBiological
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
MeasurableBiological
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
MeasurableBiological
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
Receptor, DNA,ProteinInteractions
Macro-Molecular Interactions
Cellular
NRC Toxicological Pathway
Biological Responses
THE ADVERSE OUTCOME PATHWAY
Toxicant
Molecular Initiating
Event
Chemical Reactivity
Profiles
Chemical Reactivity
Profiles
Gene Activation
Protein Production
Signal Alteration
Gene Activation
Protein Production
Signal Alteration
Receptor, DNA,ProteinInteractions
Receptor, DNA,ProteinInteractions
Macro-Molecular Interactions
Cellular Organ
Mechanistic Profiling
Biological Responses
THE ADVERSE OUTCOME PATHWAY
Toxicant
Molecular Initiating
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
Receptor, DNA,ProteinInteractions
Receptor, DNA,ProteinInteractions
Macro-Molecular Interactions
Cellular Organ
Mechanistic Profiling
In VivoTesting
Biological Responses
THE ADVERSE OUTCOME PATHWAY
Toxicant Organism
Molecular Initiating
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
Receptor, DNA,ProteinInteractions
Receptor, DNA,ProteinInteractions
Macro-Molecular Interactions Population
MAJOR PATHWAYS FOR REACTIVE TOXICITY FROM MODERATE ELECTROPHILES
Systemic Responses
SkinLiverLung
Systemic Responses
SkinLiverLung
MichaelAddition
Schiff baseFormation
SN2
Acylation
MichaelAddition
Schiff baseFormation
SN2
Acylation
AtomCentered
Irreversible(Covalent)Binding
AtomCentered
Irreversible(Covalent)Binding
InteractionMechanisms
MolecularInitiatingEvents Exposed
SurfaceIrritation
ExposedSurface
Irritation
SystemicImmune
Responses
SystemicImmune
Responses
Necrosis:Which
Tissues?
In vivoEndpoints
Pr-S AdductsGSH OxidationGSH DepletionNH2 AdductsRN AdductsDNA Adducts
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.
Adverse Outcome PathwayER-mediated Reproductive ImpairmentMeasurements across levels of biological organization
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.
Adverse Outcome PathwayER-mediated Reproductive ImpairmentMeasurements across levels of biological organization
In vivo
TISSUE/ORGAN INDIVIDUAL
Skewed Sex
Ratios;
Yr Class
POPULATION
Liver CellsAltered Protein
Expression(marker)(effect)
Vitellogenin
Liver Altered gene products
(timing, amt)
Gonad Ova-testis; Sex-reversed; Fecundity
Sex reversal;
Altered behavior;
Repro.
Adverse Outcome PathwayER-mediated Reproductive ImpairmentMeasurements across levels of biological organization
In vivo
CELLULARResponse
TISSUE/ORGAN INDIVIDUAL
Skewed Sex
Ratios;
Yr Class
POPULATION
ReceptorBinding
ER-Chemical Binding
Liver CellsAltered Protein
Expression(marker)(effect)
Vitellogenin
Liver Altered gene products
(timing, amt)
Gonad Ova-testis; Sex-reversed; Fecundity
Sex reversal;
Altered behavior;
Repro.
Adverse Outcome PathwayER-mediated Reproductive ImpairmentMeasurements across levels of biological organization
In vivo
MOLECULAR Target
CELLULARResponse
TISSUE/ORGAN INDIVIDUAL
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
TISSUE/ORGAN INDIVIDUAL
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
Reduced excitatory transmission
Facilitated inhibitory 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
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
Reduced excitatory transmission
Facilitated inhibitory 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
Amnesia Pathway for Isoflurane 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
Reduced excitatory transmission
Facilitated inhibitory transmission
Facilitated inhibitory transmission
Cortex - Thalamus
Brain Stem
Spinal Cord
Unconsciousness Loss of perceptual
awareness
Heavy Sedation Slow responses
Immobility Loss of pain
response
Kinetics Dynamics
Increasin
g Depth
of An
esthesia
Effectopedia
Cause Link Effect
Pathways for Reactive Toxicity
MichaelAddition
Schiff baseFormation
SN2
Acylation
AtomCentered
Irreversible(Covalent)Binding
InteractionMechanisms
MolecularInitiatingEvents
Membrane Alteration
___
Oxidative Stress
___
Genotoxicity
Death
ImpairedGrowth
Impaired Development
Impaired Reproduction
In vivoEndpoints
Pr-S AdductsGSH OxidationGSH DepletionNH2 AdductsRN AdductsDNA Adducts
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
Schiff baseFormation
SN2
Acylation
Atom Centered Irreversible(Covalent)
ProteinBinding
InteractionMechanisms
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
Schiff baseFormation
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
Schiff baseFormation
SN2
Acylation
AtomCentered
Irreversible(Covalent)Binding
InteractionMechanisms
MolecularInitiatingEvents Exposed
SurfaceIrritation
SystemicImmune
Responses
NecrosisWhich
Tissues?
In vivoEndpoints
Pr-S AdductsGSH OxidationGSH DepletionNH2 AdductsRN AdductsDNA Adducts
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