Yin- tak Woo, Ph.D., DABT Risk Assessment Division Office of Pollution Prevention & Toxics

1. Brief Overview of the Major Mechanisms of Nongenotoxic/Epigenetic Carcinogens and Exploration of Possible (Q)SAR Approaches Yin-tak Woo, Ph.D., DABT Risk Assessment Division Office of Pollution Prevention & Toxics U.S. Environmental Protection Agency Washington, DC 20460 Presented at 2ndMcKim Int. QSAR Workshop Baltimore, MD May 8 – 10, 2012 *Disclaimer: The views expressed are solely the author’s and do not necessarily reflect the views and policies of the U.S. EPA.

2. Outline • Nongenotoxic/Epigenetic Carcinogens Defined • Importance of Identifying Nongenotoxic Carcinogens • Difficulties of Studying Nongenotoxic Carcinogens • Major Mechanisms of Nongenotoxic Carcinogens • Possible (Q)SAR Approaches • Overview of Selected Mechanisms and (Q)SAR • Integrative approaches • Conclusions and Recommendations

3. Defining Nongenotoxic/Epigenetic Carcinogens • Carcinongesis is a multistage/multistep process • Initiation: Mutation converts normal to preneoplastic cells • Promotion: Expansion of preneoplastic cells to benign tumors • Progression: Transformation of benign to invasive malignant tumors • A complete carcinogen acts on all three stages • Classification usually based on predominant mechanism • Genotoxic carcinogens, mostly DNA-reactive, act directly on initiation as the/a predominant mechanism • Nongenotoxic carcinogens, act directly on promotion &/or progression; clonally expand previously initiated cells or trigger pathways to generate indirect genotoxic effects

4. Initiation Promotion Progression Main event(s) Direct DNA binding Indirect DNA damage Clonal expansion Cell proliferation Apoptosis Differentiation Homeostasis Overcoming suppressions (e.g., p53, immune, angiogenesis) Key mechanistic consideration Electrophile, resonance stabilization, nature of DNA adduct Receptor, cytotoxicity, gene expression Free radical, receptor, gene suppression Signal transduction, homeostasis SAR/QSAR mechanistic descriptors Electrophilicity, HOMO/LUMO, delocalization energies, …… 2D, 3D, docking, biopersistence, methylation, …. Reduction potential, 2D, 3D, ……

5. Importance of Identifying and Thoroughly Understanding Nongenotoxic Carcinogens • New generation products: avoid genotoxic • Quantitative risk assessment: threshold or not, conditional scenarios • Human relevance/significance • Regulatory impact • Testing strategies for cancer bioassay: proactive, prioritization, surrogate, weight of evidence • Molecular biology of cancer • Chemoprevention and treatment of cancer

6. Difficulties of Nongenotoxic Carcinogens • Often target organ-, species/strain-, gender-, route-, dose-, &/or exposure scenario-specific • May involve multiple mechanisms/pathways • Often indirectly genotoxic • Some genotoxic chemicals can act via nongenotoxic mechanisms under some scenarios or exposure conditions • Substantial biological understanding needed • May require confirmatory biological studies, esp. for regulation

7. (Q)SAR of nongenotoxic carcinogens • Recently one of the most active, high incentive research areas • Multiple mechanisms with no obvious unifying concept • Often target organ-specific or cell-specific • May be > 1 molecular initiating event or pathway • (Q)SAR analysis possible for some of the mechanisms • Some mechanisms may not be of human significance • Mechanism-specific predictive biological assays may provide supportive or confirmatory evidence • TXG, HTS, AOP assays and Tox-21, ToxCast strategies may provide additional input

8. Major nongenotoxic mechanisms • Receptor-mediated mechanisms (AhR, PPAR, CAR, PXR, AR, ER, other hormonal, growth factors…) • Hormonal imbalance (thyroid, testicular, mammary, ovary,..) • Cytotoxicity-induced cell proliferation (often organ specific) • Oxidative Stress (ROS) and Nitrosative Stress (RNS) • Inhibition of Intercellular Communication (GJIC) • Perturbation of DNA methylation/gene expression • Miscellaneous (phosphatase inhibition, choline deficiency, immunosuppression, spindle poison, apoptosis, signal transduction, angiogenesis, etc.)

9. Receptor-Mediated MechanismsGeneral (Q)SAR Approaches • Receptor-specific (e.g., nuclear vs. membrane vs protein; well defined vs. promiscuous) • Molecular size and shape (2D vs. 3D) • Some may have critical regions, active site • Mostly reversible binding • Biological persistence of the chemical/ligand • Measure biological half-life of the chemical/ligand • Structural features of metabolic refractoriness

10. TCDD Ah Receptor Agonists SAR *CE = clear evidence; SE = some evidence; EE = equivocal evidence **Highest incidence observed in any one specific target organ.

11. Peroxisome Proliferator-Activated Receptor (PPARα) SAR

12. Relative peroxisome proliferative activity of chlorinated phenoxyacetic acid in cultured hepatocytes

13. PPARα carcinogens: SAR and biological features • Medium size chemicals with polar and nonpolar ends • Polar end being carboxylic acid moiety in most cases • Nonpolar end may be a variety of chemical structures (e.g., branched alkyl [omega minus 1]; polyhalogenated alkyl [esp. perfluoro]; ring-substituted phenoxy, etc.) • Organ-, species/strain-, gender- specific • Hepatocarcinogenicity correlates with peroxisome proliferative activity • Biological half-life can be an important factor due to noncovalent binding • Metabolic refractoriness may be important factor

14. CAR and PXR LigandsOmiecinski et al. Toxicol. Sci. 120 (S1), S49-S75, 2011

15. Hormonal imbalance: basic principles

16. Hormonal imbalance: (Q)SAR approaches • Precursor transport modifiers (e.g., ↓ iodide by perchlorate) • Biosynthetic pathway modifiers (e.g., ↓ thyroid peroxidase by Amitrole, PTU; ↓ 5’-monodeiodonase by red dye no.2) • Catabolism of hormone by P450 inducers (e.g., phenobarbital, TCDD) • Hormone secretion (e.g., ↓ thyroid by lithium?)

17. Some SA for thyroid carcinogens

18. Oxidative stress of nongenotoxic carcinogens • Structural variety: quinones and quinoids, aromatic amines, polyhalogenated hydrocarbons, oxidants, transition metal compounds, peroxy compounds, etc. • Free radicals, reactive oxygen species, lipid peroxides, malondialdehyde, etc. as secondary reactants • Free radical stabilization may be a factor for some (SAR) • 8-Hydroxy/8-Oxo-2’-deoxyguanosine an important biomarker for indirect DNA damage • Lipid peroxides and malondialdehydes measurable by thiobarbituric acid (TBA) assay • Antioxidants and free radical scavengers protective

19. Formation of 8-oxo-dG and 8-OHdG in oxidative stress(from Valavanidis et al. J. Env.Sci. Hlth. C27, 120, 2009)

20. Intercellular Communication (GJIC) Inhibitionas a Nongenotoxic carcinogenic mechanism CF3(CF2)nCOOH n= 0 to 3,14,or 16, inactive; n= 4, weak; n=5-8, active CF3(CF2)nSO3H n=3, inactive; n=5 or 7, active

21. Perturbation of DNA methylation/gene expression • Methylation of cytosine at 5-position regulates gene expression • Altered DNA methylation may lead to carcinogenesis • Global vs. regional • Alteration of methyl donor (SAM) pool (e.g., ↓ As, 5-azadeoxycytidine; ↑ methionine, choline, etc.) • Manipulation of methyltransferase • Microarray-based transcriptional studies

22. Cytotoxicity-induced regenerative cell proliferation • Male rat kidney tumors via α2µnephropathy (e.g., tert-butyl alcohol, jet fuels, unleaded gasoline) • Bladder stones, calculi, microcrystals (e.g., NTA, saccharin, melamine, sulfonamides, Fosetyl) • Liver tumors via “chemical hepatectomy” (e.g., carbon tetrachloride, chloroform) • Spleen tumors via splenotoxicity/hemosiderosis (e.g., hemolytic aromatic amines and azodyes) • Nasal tumors via in situ formation of reactive chemicals or acids (e.g., Alachlor, vinyl acetate) • Persistent portal of entry (e.g., lung, skin) exposure to acids or irritants (e.g., sulfuric acid mist, petrol distillates)

23. Cytotoxicity via Alpha-2µ NephropathyCriteria for classification • The renal tumors occur only in male rats • Acute exposure cause hyaline droplet in proximal convoluted tubules (P2-segment) • Protein in hyaline droplet = α2µ-globulin • Observation of Hallmark histopath lesions (granular casts, linear papillary mineralization) • Absence of hyaline droplets and histopath in female rats and mice of both sexes • Negative in short term tests for genotoxicity

24. Searching for a common mechanistic basis

25. Small tert-alcohol as potential SA formale rat kidney α2µnephropathy

26. Cytotoxicity induced nasal tumors

27. Cytotoxicity Induced: hepatotoxicity via Inhibition of Protein Phosphates 1, 2A (from Dr. Fujiki’s lab)

28. Microcystin-LR: chemical structure and SAR(red: Adda region; green : 6(Z)- stereoisomer)

29. Binding of Okadaic Acid and Microcystin-LRto Protein Phosphate 2A Core Enzyme (from Xing et al., Cell 127, 341, 2006)

30. Integrative Approach Initiation (e.g., Electrophilicity, DNA adduct, genotox, transgenic rodent models, ras, etc.) Promotion (e.g, cell proliferation, apoptosis, gap junct. cyp, hormonal imbal, gene expression, mitogenesis, ppar, myc, etc.) Progression (e.g, immune suppression, free radical, metastasis, angiogenesis, etc.) Woo et al. (1998)

31. EPA gene expression and pathway analysis study of nongenotoxic carcinogenic conazole pesticides(from Hester et al. Tox. Sci. 127, 54, 2012)

32. Highlights from the study • Microarray-based transcriptional analysis showed a common basis • 330 common altered genes for Cyp, GSH S-transferase and oxidative stress • Subset of 80 altered genes associated with cancer. • Pathways associated: xenobiotic metabolism, oxidative stress, cell signaling, and cell proliferation. • Common TGFa-centric pathway provides a more refined toxicity profile

33. TGFα-mediated cell proliferation as the commonmechanistic pathway for Cypro, Epoxi and Propi

34. Cancer Hallmark processes* and pathways** • Inducing Angiogenesis (VEGF, IGF-1R, miRNA) • Resisting cell death (apoptosis, p53, DNA damage) • Sustaining proliferative signaling (growth factors, Akt, MAP kinase, oncogenes) • Evading growth suppressors (p53, apoptosis, TGFβ, EMT) • Enabling cell immortality (telomerase, p53,.DNA repair) • Activating invasion and metastasis (EMT, tumor metastasis) • Evading Immune destruction (T- & B-cell, inflammatory response) • Reprogramming energy metabolism (hypoxia, glycosylation) • *Hanahan and Weinberg, Cell, 144,646 (2011). **see “Pathway Central” online. • EPA ToxCast study mapping assays to cancer hallway processes ongoing

35. Suggestions for (Q)SAR of Nongenotoxic carcinogens • Expansion of database (e.g., OPP, pharma) • Structural and Functional Classifications • Structural alerts and modification factors • Integrating with HTS, TXG, pathway analysis • Defining conditions and scenarios • Building consensus and testing strategies • Beware of real life exposure and susceptible subpopulations