McKim Conference on Predictive Toxicology The Inn of Lake Superior Duluth, Minnesota

1. Toxicity Pathways as an Organizing Concept Gilman Veith McKim Conference on Predictive Toxicology The Inn of Lake Superior Duluth, Minnesota September 16-18, 2008

2. Summary of Past Discussions • Need for a Paradigm Shift – Kees, Bradbury • Need to Integrate Toxicology – Veith • Soft Reactive Toxicity – Schultz, Comber • Need for Organizing Chemistry – Mekenyan • ReproTox Pathways – Schmieder, Ankley … • NeuroTox Pathways – Fowle, Bushnell,

3. Purpose of McKim 2008 • Status on the new mindset in Europe • Progress on skin sensitisation models • Progress on the QSAR Application Toolbox • Explore organizing methods for targets • Expand the delineation of toxicity pathways

4. QSAR QSAR is the study of how to organize experiments of chemical behavior to find order Order is found when similarity is well-grounded in chemistry…”small” changes are often huge. The hypothesis-driven paradigm will require integration of QSAR with more diagnostic assays

5. Why Build QSAR Methods? Example: estimated ER Binding Affinity for 6,518 High Production Volume Chemicals RBA

6. Our Conceptual Framework Chemical Speciation and Metabolism Molecular Initiating Events Measurable Biological Effects Adverse Outcomes Parent Chemical

7. Our Conceptual Framework Speciation and Metabolism Molecular Initiating Events Measurable Biological Effects Adverse Outcomes Parent Chemical Response Pathways Chemistry/ Biochemistry QSAR 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

8. Conceptual Framework Chemical Speciation and Metabolism Molecular Initiating Events Measurable Biological Effects Adverse Outcomes Parent Chemical Mortality -systemictoxicity -disease -cancer Impaired Development -terata -prenatal deficits Reproductive Fitness -fertility -viable offspring • Interaction • Mechanisms • -Nonspecific • Targets • Atom Centers • Targets • -Receptor • Targets Chemical Inventories and Categories (~200,000)

9. Major Pathway for Reactive Toxicants To Fish Vulnerable Organ Pathology Molecular Initiating Events Interaction Mechanisms In vivo Endpoints Pathogenesis Michael Addition Schiff base Formation SN2 Acylation Atom Centered Irreversible (Covalent) Protein Binding Death from Suffocation “Any Exposed Surface” Changes Necrosis of the Gill Epithelium Complexes Membranes, etc

10. At the Molecular Initiating Event Chemical Speciation and Metabolism Molecular Initiating Events Measurable Biological Effects Adverse Outcomes Parent Chemical The QSAR Question is: “How many other chemicals can interact at this target?” While the Toxicology Question is: “What are the known biological effects from this altered target?”

11. From the Library of Initiating Events Chemical Speciation and Metabolism Library Of Molecular Initiating Events Measurable Biological Effects Adverse Outcomes Parent Chemical OECD Toolbox Chemical Profiler Handles the Chemistry for QSAR Models Conformations Targets Interactions Metabolic Simulators Inventories Structural Requirements

12. Which Metabolite should we use in modeling interactions? Simulated 2-Acetylaminofluorene Metabolism

13. Which Conformation should we use to model interactions?

14. From the Library of Initiating Events Chemical Speciation and Metabolism Library Of Molecular Initiating Events Measurable Biological Effects Adverse Outcomes Parent Chemical OECD Toolbox Libraries Will Link Targets to Adverse Effects Altered Gonad Development Gene Activation ER Binding Impaired Reproduction Protein Production

15. Pathways for Reactive Toxicity from Soft Electrophiles Mechanisms Molecular Initiating Events In vivo Endpoints Michael Addition Schiff base Formation SN2 Acylation Atom Centered Irreversible (Covalent) Protein Binding Exposed Surface Irritation Necrosis Skin Lung/Gills GI Tract No Immunogenic Systemic Immune Responses Systemic Responses Skin Liver Lung Yes

16. Major Pathways for Reactive Toxicity from Moderate Electrophiles Interaction Mechanisms Molecular Initiating Events In vivo Endpoints Exposed Surface Irritation Michael Addition Schiff base Formation SN2 Acylation Atom Centered Irreversible (Covalent) Binding Necrosis Which Tissues? Pr-S Adducts GSH Oxidation GSH Depletion NH2 Adducts RN Adducts DNA Adducts Oxidative Stress Systemic Responses Skin Liver Lung Systemic Immune Responses Dose-Dependent Effects

17. Nonpolar Narcosis Observable Response Level Nonspecific Reactive NOAEL—Long Term Receptor Off-Target Therapeutic Effects 1.0 0.01 0.1 Chemical Activity (or comparable metric)

18. Pathways for Reactive Toxicity Molecular Initiating Events In vitro Endpoints Interaction Mechanisms In vivo Endpoints Membrane Alteration _ _ _ Oxidative Stress _ _ _ Genotoxicity Death Impaired Growth Impaired Development Impaired Reproduction Michael Addition Schiff base Formation SN2 Acylation Atom Centered Irreversible (Covalent) Binding Pr-S Adducts GSH Oxidation GSH Depletion NH2 Adducts RN Adducts DNA Adducts Dose-Dependent Pathways Species/Sex/Life-Stage

19. Two Questions for Building Pathways Direct Reaction Effect #1 Pr-S Adducts GSH Oxidation GSH Depletion NH2 Adducts RN Adducts DNA Adducts Altered Synthesis Effect #2 Oxidation Effect #3 How Many Ways to Deplete GSH? How Many Downstream Effects?

20. Delineation of Toxicity Pathways Linkages Across Levels of Biological Organization In vivo Methods In vitro Methods In Silico Methods Molecular/ Subcellular Electronic Cell Tissue Organ Individual Exposure/ Metabolism Penetration Routes Detoxification Pathways Activation Pathways Chemical Reactivity Profiles Reversible Nonspecific Binding Reversible Specific Binding Covalent Binding Response Pathways Regulatory Endpoints Chemical Inventories Molecular Initiating Events Membranes Energy Charge Nuclear Receptors Protein Synthesis DNA Integrity Lethality Growth Development Reproduction More Relevant Endpoints Intrinsic Chemical Attributes Better Defined Endpoints