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Predicting Indirect DNA Damage by Simulating Metabolic Activation of Chemicals
1
2nd McKim Workshop on Reducing Data Redundancy in Cancer AssessmentBaltimore, 8-10 May 2012
Ovanes Mekenyan, Milen Todorov, Ksenia Gerova
Laboratory of Mathematical Chemistry, Bulgaria
2
Outlook
• Goal• Methods• Data• Predicting:
• AMES mutagenicity without metabolic activation• AMES metabolic activation chemicals negative as parents• Illustrating metabolic activation • False positives after metabolic activation• False negatives after metabolic activation
• Conclusions
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Outlook
• Goal• Methods• Data• Predicting:
• AMES mutagenicity without metabolic activation• AMES metabolic activation chemicals negative as parents• Illustrating metabolic activation • False positives after metabolic activation• False negatives after metabolic activation
• Conclusions
4
• Predicting indirect DNA damage in the General Workflow Diagram for screening large chemicals inventories for carcinogenicity
Goal
5
General Flow Diagram for Screening Large Inventories for carcinogenicity
Inventory
Direct DNA Reactive
Chemicals
DNA reactiveMetabolites
Ames Positive with S9
Ames Positive w/o S9
Classify asGenotoxic Bacterial Mutagen
High Carcinogenicity
Potential?
Generate metabolites
YY
Receptor-Based Screening
Low Carcinogenit
Potential
Y
N
Y Y
Protein Reactive
Chemicals
High Priority for Tumor
Promotion Assays
No-ThresholdRisk Assessment
CTA Assays for Nongenotoxic/
EpigeneticChemicals
Intermediate Priority for
Tumor Promotion
AssaysThreshold EffectRisk Assessment
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General Flow Diagram for Screening Large Inventories for carcinogenicity
Inventory
Direct DNA reactive
Indirect DNA reactive
Ames Positive with S9
Ames Positive w/o S9
Bacterial Mutagen
Chrom Ab ?MicroNucl ?
Protein OASIS
Generate metabolites
N
Y
Y
Receptor-Based Epigenetic
Screen
Low Carcinogenit
Potential
Y
Chrom Ab ?MicroNucl ?
Refine TIMES/Structural alerts
N
YY
N
Oxidative stress?
In vivo Mammal Tests
Protein Reactive
Return for further screening
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Outlook
• Goal• Methods• Data• Predicting:
• AMES mutagenicity without metabolic activation• AMES metabolic activation chemicals negative as parents• Illustrating metabolic activation • False positives after metabolic activation• False negatives after metabolic activation
• Conclusions
DNA binding profile by OASIS
DNA binding profile by OECD
8
Methods:
• QSAR Toolbox profiles for DNA binding
Illustration of the DNA binding profile of the QSAR Toolbox
9
Known DNA (covalent) binding mechanisms
Known DNA (covalent) binding mechanisms
Structural boundaries of the category
Structural boundaries of the category
DNA binding profile by OASIS
DNA binding profile by OECD
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Methods:
• QSAR Toolbox profiles for DNA binding
• TIMES Metabolic simulator for rat liver S9
OASIS Metabolic Simulator
• Prioritized list of non-enzymatic (abiotic) and enzymatic molecular transformations;
• Molecular transformations are characterized by:
Source and product fragments;Inhibiting “masks” preventing the
application of metabolic reactions if necessary;
• Substructure-matching software engine applies the simulated biochemical
• Reproduces the documented metabolic pathways and toxicity endpoint resulting from metabolic activation of chemicals
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Illustration the OASIS Metabolic Simulators
(extract from the Rat in vivo metabolism simulator)
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Substrate Principle transformations MetabolitesSimulator of metabolismSimulator of metabolism
Aliphatic C-oxidation
C CH3 C CH2OH
Epoxidation
C C C C
O
Aliphatic C-oxidation
C CH2OH C C
O
H
Epoxide Hydration
C C
O
C C OHHO
Aliphatic C-oxidation
C C
O
HC C
O
OH
O-Glucuronidation
C OH C O
O
HOH H
OH
H
OHH
COOH
H
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Substrate Principle transformations MetabolitesSimulator of metabolismSimulator of metabolism
Aliphatic C-oxidation
C CH3 C CH2OH
Epoxidation
C C C C
O
Aliphatic C-oxidation
C CH2OH C C
O
H
Epoxide Hydration
C C
O
C C OHHO
Aliphatic C-oxidation
C C
O
HC C
O
OH
O-Glucuronidation
C OH C O
O
HOH H
OH
H
OHH
COOH
H
P= 0.90
P= 0.93
P= 0.94
P= 0.95
P= 0.96
P= 0.97
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Substrate Principle transformations Metabolites
Aliphatic C-oxidation
C CH3 C CH2OH
Epoxidation
C C C C
O
Aliphatic C-oxidation
C CH2OH C C
O
H
Epoxide Hydration
C C
O
C C OHHO
Aliphatic C-oxidation
C C
O
HC C
O
OH
O-Glucuronidation
C OH C O
O
HOH H
OH
H
OHH
COOH
H
P= 0.94
P= 0.93
P= 0.90
P= 0.95
P= 0.96
P= 0.97
CH2C CH3
- Isopropenylbenzene
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Match? - No! C CH2OH C CO
H
Substrate Principle transformations Metabolites
Aliphatic C-oxidation
C CH3 C CH2OH
Epoxidation
C C C C
O
C C
O
C C OHHO
Aliphatic C-oxidation
C C
O
HC C
O
OH
O-Glucuronidation
C OH C O
O
HOH H
OH
H
OHH
COOH
H
P= 0.94
P= 0.93
P= 0.90
P= 0.95
P= 0.96
Aliphatic C-oxidationCH2C CH3
P= 0.97
Epoxide Hydration
- Isopropenylbenzene
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C CH2OH C C
O
H
Substrate Principle transformations Metabolites
P= 0.97
Aliphatic C-oxidation
C CH3 C CH2OH
Epoxidation
C C C C
O
C C
O
C C OHHO
Aliphatic C-oxidation
C C
O
HC C
O
OH
O-Glucuronidation
C OH C O
O
HOH H
OH
H
OHH
COOH
H
P= 0.94
P= 0.93
P= 0.90
P= 0.95
CH2C CH3
P= 0.96
Aliphatic C-oxidation
Epoxide Hydration
- Isopropenylbenzene
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Match? - No! C C
O
HO C C OH
Substrate Principle transformations Metabolites
C CH2OH C C
O
H
Aliphatic C-oxidation
C CH3 C CH2OH
Epoxidation
C C C C
O
Aliphatic C-oxidation
C C
O
HC C
O
OH
C OH C O
O
HOH H
OH
H
OHH
COOH
H
P= 0.94
P= 0.95
CH2C CH3
P= 0.93
P= 0.90
O-Glucuronidation
P= 0.96
Aliphatic C-oxidation
Epoxide Hydration
P= 0.97
- Isopropenylbenzene
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Substrate Principle transformations Metabolites
C C
O
C C
Epoxidation
RESULTMatch? - Yes!C
H2CCH3
OC
H2C CH3
P= 0.95
C CH2OH C C
O
H
Aliphatic C-oxidation
C C
O
C C OHHOP= 0.96
Epoxide Hydration
Aliphatic C-oxidation
C CH3 C CH2OH
Aliphatic C-oxidation
C C
O
HC C
O
OH
O-Glucuronidation
C OH C O
O
HOH H
OH
H
OHH
COOH
H
P= 0.94
P= 0.93
P= 0.90
P= 0.97
Generated map
1.1
Epoxidation
- Isopropenylbenzene
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Aliphatic C-oxidation
Substrate Principle transformations Metabolites
RESULTMatch? - Yes!
CH2C CH3
C CH3 C CH2OH
CC CH2OHH2
P= 0.94
C CH2OH C C
O
H
Aliphatic C-oxidation
P= 0.96
Epoxide Hydration
C C
O
C C OHHO
Epoxidation
C C C C
O
P= 0.95
Aliphatic C-oxidation
C C
O
HC C
O
OH
C OH C O
O
HOH H
OH
H
OHH
COOH
H
P= 0.93
P= 0.90
O-Glucuronidation
P= 0.97
- Isopropenylbenzene
1.1
C-oxidation
1.2
Generated map
Epoxidation
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Aliphatic C-oxidation
C C
O
HC C
O
OH
O-Glucuronidation
C OH C O
O
HOH H
OH
H
OHH
COOH
H
C CH2OH C C
O
H
Epoxide Hydration
Aliphatic C-oxidation
Aliphatic C-oxidation
C C
O
C C OHHO
Substrate Principle transformations Metabolites
C C
O
C C
Epoxidation
Match?
CH2C CH3
C CH3 C CH2OH
- No!
P= 0.94
P= 0.93
P= 0.90
P= 0.95
P= 0.96
P= 0.97
Generated map
Epoxidation C-oxidation
- Isopropenylbenzene
1.1 1.2
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Aliphatic C-oxidation
C C
O
HC C
O
OH
O-Glucuronidation
C OH C O
O
HOH H
OH
H
OHH
COOH
H
C CH2OH C C
O
H
Epoxide Hydration
Aliphatic C-oxidation
Aliphatic C-oxidation
C C
O
C C OHHO
Substrate Principle transformations Metabolites
C C
O
C C
Epoxidation
Match?
CH2C CH3
- No!
C CH3 C CH2OHP= 0.94
P= 0.93
P= 0.90
P= 0.95
P= 0.96
P= 0.97
Generated map
Epoxidation C-oxidation
- Isopropenylbenzene
1.1 1.2
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Aliphatic C-oxidation
C C
O
HC C
O
OH
O-Glucuronidation
C OH C O
O
HOH H
OH
H
OHH
COOH
H
C CH2OH C C
O
H
Epoxide Hydration
Aliphatic C-oxidation
Aliphatic C-oxidation
C C
O
C C OHHO
Substrate Principle transformations Metabolites
C C
O
C C
Epoxidation
Match? - No!
C CH3 C CH2OHP= 0.94
P= 0.93
P= 0.90
P= 0.95
P= 0.96
P= 0.97
Generated map
Epoxidation C-oxidation
1.1 1.2
CH2C
OCH3
- Metabolite 1.1
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Aliphatic C-oxidation
C C
O
HC C
O
OH
O-Glucuronidation
C OH C O
O
HOH H
OH
H
OHH
COOH
H
C CH2OH C C
O
H
Epoxide Hydration
Aliphatic C-oxidation
Aliphatic C-oxidation
Substrate Principle transformations Metabolites
C C
O
C C
Epoxidation
Match?- Yes!
C CH3 C CH2OHP= 0.94
P= 0.93
P= 0.90
P= 0.95
P= 0.96
P= 0.97
CH2C
OCH3
C C
O
HO C C OH RESULT
C OH
OH
CH2
1.1 1.2
2.1
Hydration
C-oxidationEpoxidation
Generated map- Metabolite 1.1
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Aliphatic C-oxidation
C C
O
HC C
O
OH
O-Glucuronidation
C OH C O
O
HOH H
OH
H
OHH
COOH
H
Epoxide Hydration
Aliphatic C-oxidation
Aliphatic C-oxidation
Substrate Principle transformations Metabolites
C C
O
C C
Epoxidation
Match?- Yes!
C CH3 C CH2OHP= 0.94
P= 0.93
P= 0.90
P= 0.95
P= 0.96
P= 0.97
1.1 1.2
RESULTC OH
OH
CH2
- Metabolite 2.1
C C
O
C C OHHO
C CH2OH C CO
H
C OH
O
CH2
1.1 1.2
2.1
Generated map
Epoxidation C-oxidation
3.1
C-oxidation
Hydration
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Aliphatic C-oxidation
C C
O
HC C
O
OH
O-Glucuronidation
C OH C O
O
HOH H
OH
H
OHH
COOH
H
Epoxide Hydration
Aliphatic C-oxidation
Aliphatic C-oxidation
Substrate Principle transformations Metabolites
C C
O
C C
Epoxidation
Match?- Yes!
C CH3 C CH2OHP= 0.94
P= 0.93
P= 0.90
P= 0.95
P= 0.96
P= 0.97
1.1 1.2
RESULT
C C
O
C C OHHO
C CH2OH C CO
H 1.1 1.2
2.1
Generated map
Epoxidation C-oxidation
3.1
C-oxidation
Hydration
- Metabolite 1.2.
CC CH2OHH2 O
C
HCCH2
C-oxidation2.2
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Aliphatic C-oxidation
C C
O
HC C
O
OH
O-Glucuronidation
C OH C O
O
HOH H
OH
H
OHH
COOH
H
Epoxide Hydration
Aliphatic C-oxidation
Aliphatic C-oxidation
Substrate Principle transformations Metabolites
C C
O
C C
Epoxidation
Match?- No!
C CH3 C CH2OHP= 0.94
P= 0.93
P= 0.90
P= 0.95
P= 0.96
P= 0.97
1.1 1.2
C C
O
C C OHHO
1.1 1.2
2.1
Generated map
Epoxidation C-oxidation
3.1
C-oxidation
Hydration
- Metabolite 2.2.
OC
HCCH2
C-oxidation2.2
C CH2OH C C
O
H
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Aliphatic C-oxidation
C C
O
HC C
O
OH
O-Glucuronidation
C OH C O
O
HOH H
OH
H
OHH
COOH
H
Epoxide Hydration
Aliphatic C-oxidation
Aliphatic C-oxidation
Substrate Principle transformations Metabolites
C C
O
C C
Epoxidation
Match?- No!
C CH3 C CH2OHP= 0.94
P= 0.93
P= 0.90
P= 0.95
P= 0.96
P= 0.97
1.1 1.2
C C
O
C C OHHO
1.1 1.2
2.1
Generated map
Epoxidation C-oxidation
3.1
C-oxidation
Hydration
OC
HCCH2
C-oxidation2.2
C CH2OH C C
O
H
- Metabolite 2.2.
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Aliphatic C-oxidation
C C
O
HC C
O
OH
O-Glucuronidation
C OH C O
O
HOH H
OH
H
OHH
COOH
H
Epoxide Hydration
Aliphatic C-oxidation
Aliphatic C-oxidation
Substrate Principle transformations Metabolites
Epoxidation
Match?- Yes!
C CH3 C CH2OHP= 0.94
P= 0.93
P= 0.90
P= 0.96
P= 0.97
1.1 1.2
C C
O
C C OHHO
1.1 1.2
2.1
Generated map
Epoxidation C-oxidation
3.1
C-oxidation
Hydration
OC
HCCH2
C-oxidation2.2
C CH2OH C C
O
H
C C
O
C C
(Conjugated aldehyde group prevents epoxidation)
- Metabolite 2.2.
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Aliphatic C-oxidation
C C
O
HC C
O
OH
O-Glucuronidation
C OH C O
O
HOH H
OH
H
OHH
COOH
H
Epoxide Hydration
Aliphatic C-oxidation
Aliphatic C-oxidation
Substrate Principle transformations Metabolites
Epoxidation
Match?- No! C CH3 C CH2OHP= 0.94
P= 0.93
P= 0.90
P= 0.96
P= 0.97
1.1 1.2
C C
O
C C OHHO
1.1 1.2
2.1
Generated map
Epoxidation C-oxidation
3.1
C-oxidation
Hydration
OC
HCCH2
C-oxidation2.2
C CH2OH C C
O
H
C C
O
C CP= 0.95
- Metabolite 2.2.
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Aliphatic C-oxidation
O-Glucuronidation
C OH C O
O
HOH H
OH
H
OHH
COOH
H
Epoxide Hydration
Aliphatic C-oxidation
Aliphatic C-oxidation
Substrate Principle transformations Metabolites
Epoxidation
Match?- Yes!
C CH3 C CH2OHP= 0.94
P= 0.93
P= 0.90
P= 0.96
P= 0.97
1.1 1.2
C C
O
C C OHHO
1.1 1.2
2.1
Generated map
Epoxidation C-oxidation
3.1
C-oxidation
Hydration
OC
HCCH2
C-oxidation2.2
C CH2OH C C
O
H
C C
O
C CP= 0.95
C C
O
H
C C
O
OH
RESULT
OC
CCH2
OH
3.2
C-oxidation
- Metabolite 2.2.
Metabolic SimulatorsBridging the “Parent Gap”
Virtual metabolism uses a heuristic substructure search engine applied to a hierarchy of possible molecular transformations
Library ofBiotransformations& Abiotic Reactions
Documented Partial Maps
Algorithm for optimizingTransformationProbabilities
(Rate constants)
MetabolicMaps and ReactivityProfiles
Metabolic Simulators
ParentChemicals
Simulated Metabolic Activation of 2-AcetylaminofluoreneSimulated Metabolic Activation of 2-Acetylaminofluorene(AMES mutagenicity in Rat liverS9)(AMES mutagenicity in Rat liverS9)
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
Documented
The OASIS Simulators of Mammalian Metabolism
•Liver S9 metabolism
•Different level of biological organisms (US EPA)Rat liver subcellular (microsomal)Rat liver cellular (in vitro)Organism (in vivo)
•In vivo metabolism – rat liver (in vivo MNT)In vivo detoxification logicIn vivo bioactivation
•Skin metabolism