1. QSAR in CANCER ASSESSMENT PURPOSE and AGENDA Gilman Veith Duluth MN May 19-21, 2010

2. Lethality Sensitization Birth Defect Reproductive Impairment Cancer Altered Function Altered Development Gene Activation Protein Production Signal Alteration Chemical Reactivity Profiles Receptor, DNA, Protein Interactions Structure Extinction Cellular Organ Mechanistic Profiling In Vivo Testing Biological Responses ITS and Adverse Outcome Pathway T oxicant Organism Macro -Molecular Interactions Molecular Initiating Event Population Cellular & In Vitro Testing

3. PATHWAYS IN REACTIVE CHEMICALS Michael Addition Schiff base Formation S N 2 Acylation Michael Addition Schiff base Formation S N 2 Acylation Atom Centered Irreversible (Covalent) Binding Atom Centered Irreversible (Covalent) Binding Interaction Mechanisms Molecular Initiating Events In vivo Endpoints Pr-S Adducts GSH Oxidation GSH Depletion NH2 Adducts RN Adducts DNA Adducts Pr-S Adducts GSH Oxidation GSH Depletion NH2 Adducts RN Adducts DNA Adducts In vitro Endpoints Death Impaired Growth Impaired Development Impaired Reproduction Cancer Membrane Alteration _ Oxidative Stress _ Genotoxicity

4. OXIDATIVE STRESS from GSH DEPLETION Pr-S Adducts GSH Oxidation GSH Depletion NH2 Adducts RN Adducts DNA Adducts Oxidative Stress Cell toxicity Other Effects Direct GSH Reactions Altered Synthesis Oxidation How Many Ways to Deplete GSH?How Many Downstream Effects?

5. Karlberg et al. Chem. Res. Toxicol. 2008, 21, 53-69. 1. Haptenation; 2. Epidermal inflammation & LC activation; 3. LC migration; 4. DC: T cell interaction; 5. T cell proliferation; 6. Increase in hapten-specific T cells; 7. Hapten re-exposure; 8. Acute inflammation; 9. T cell-mediated inflammation

6. GROUPING BY CANCER PATHWAYS  Genotoxic Carcinogenesis  Direct DNA damage through abiotic chemical “binding”  Most electrophiles bind to many DNA/protein sites  Metabolic differences impact cell, organ, species sensitivity  Epigenetic Carcinogenesis  Cytoxicity-induced cell proliferation  Receptor-mediated pathways  Disturbance of homeostatic control  Loss of immune surveillance  Oxidative Stress- Indirect DNA damage  Loss of intercellular communication

7. Mode of Action Receptor-mediated pathways Disturbance of homeostaticcontrol Loss of immune surveillance Oxidative Stress- Indirect DNA damage Loss of intercellular communication Cytotoxicity-induced cell proliferation GENOTOXICITY Direct DNA Damage Indirect DNA Damage NON-GENOTOXICITY

8. Receptor-mediated pathways Disturbance of homeostaticcontrol Loss of immune surveillance Oxidative Stress- Indirect DNA damage Loss of intercellular communication Cytotoxicity-induced cell proliferation GENOTOXICITY Direct DNA Damage Indirect DNA Damage NON-GENOTOXICITY DNA Mechanism #1 DNA Mechanism #2 DNA Mechanism #3 DNA Mechanism #n …………………………… Prot Mechanism #1 Prot Mechanism #2 Prot Mechanism #3 …………………………… Prot Mechanism #n AR Binding ER Binding Aromatase Inh. Thyroid Disturbance Mechanism/AlertMode of action

9. Receptor-mediated pathways Disturbance of homeostaticcontrol Loss of immune surveillance Oxidative Stress- Indirect DNA damage Loss of intercellular communication Cytotoxicity-induced cell proliferation GENOTOXICITY Direct DNA Damage Indirect DNA Damage NON-GENOTOXICITY Mechanism/Alert DNA #1 DNA #2 DNA #3 DNA #n …………. Prot #1 Prot #2 Prot #3 ………….. Prot #n AR Binding ER Binding Aromatase Inh. Thyroid Disturbance Mechanism/Metabolites DNA Mechanism #1-Met 1…m DNA Mechanism #2-Met 1…m DNA Mechanism #3-Met 1…m …………………………… DNA Mechanism #3-Met 1…m Prot Mechanism #1 - Met 1..m Prot Mechanism #2 - Met 1..m Prot Mechanism #3 - Met 1..m …………………………… Prot Mechanism #n - Met 1..m Receptor-mediated pathways -Met 1…m Disturbance of homeostatic control -Met 1…m Loss of immune surveillance-Met 1…m Oxidative Stress- Indirect DNA damage -Met 1…m Loss of intercellular comm.-Met 1…m Cytotoxicity-induced cell proliferation-Met 1…m Mode of action

10. Receptor-mediated pathways Disturbance of homeostaticcontrol Loss of immune surveillance Oxidative Stress- Indirect DNA damage Loss of intercellular communication Cytotoxicity-induced cell proliferation GENOTOXICITY Direct DNA Damage Indirect DNA Damage NON-GENOTOXICITY Mechanism/Alert DNA #1 DNA #2 DNA #3 DNA #n …………. Prot #1 Prot #2 Prot #3 ………….. Prot #n AR Binding ER Binding Aromatase Inh. Thyroid Disturbance Mechanism/Metabolites Receptor-mediated pathways -Met 1…m Disturbance of homeostatic control -Met 1…m Loss of immune surveillance-Met 1…m Oxidative Stress- Indirect DNA damage -Met 1…m Loss of intercellular comm.-Met 1…m Cytotoxicity-induced cell proliferation-Met 1…m DNA #1-Met 1…m DNA #2-Met 1…m …………………………… DNA #3-Met 1…m Prot #1 - Met 1..m Prot #2 - Met 1..m Prot #3 - Met 1..m …………………………… Prot #n - Met 1..m DNA #3-Met 1…m In vitro data AmesAmes+S9CA CA+S9 MLAMLA+S9CTA Mode of action

11. Receptor-mediated pathways Disturbance of homeostaticcontrol Loss of immune surveillance Oxidative Stress- Indirect DNA damage Loss of intercellular communication Cytotoxicity-induced cell proliferation GENOTOXICITY Direct DNA Damage Indirect DNA Damage NON-GENOTOXICITY Mechanism/Alert DNA #1 DNA #2 DNA #3 DNA #n …………. Prot #1 Prot #2 Prot #3 ………….. Prot #n AR Binding ER Binding Aromatase Inh. Thyroid Disturbance Mechanism/Metabolites Receptor-mediated pathways -Met 1…m Disturbance of homeostatic control -Met 1…m Loss of immune surveillance-Met 1…m Oxidative Stress- Indirect DNA damage -Met 1…m Loss of intercellular comm.-Met 1…m Cytotoxicity-induced cell proliferation-Met 1…m DNA #1-Met 1…m DNA #2-Met 1…m …………………………… DNA #3-Met 1…m Prot #1 - Met 1..m Prot #2 - Met 1..m Prot #3 - Met 1..m …………………………… Prot #n - Met 1..m DNA #3-Met 1…m In vitro data Ames Ames+S9 CA CA+S9 MLA MLA+S9 CTA In vivo data COMETUDSCAMN(BN)CTARCA Mode of action

12. SCREENING LEVEL HAZARD ID Direct DNA Binding Nongenotoxic Mechanisms Activated Metabolites Indirect DNA Damage Parent Chemical Individual Initiating Events/ Structural Alerts Grouping Data for Interaction Categories Structural Evidence of Cancer Potential Category with Data for Cancer Potential No Evidence Of Cancer Potential