Brief Overview of the Major Mechanisms of Nongenotoxic/Epigenetic Carcinogens and Exploration of Possible (Q)SAR Approaches

Yin-tak Woo, Ph.D., DABTRisk Assessment Division

Office of Pollution Prevention & ToxicsU.S. Environmental Protection Agency

Washington, DC 20460

Presented at 2nd McKim Int. QSAR WorkshopBaltimore, 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.

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

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

  Initiation Promotion Progression

Main event(s) Direct DNAbindingIndirect DNAdamage

Clonal expansion Cell proliferation Apoptosis Differentiation Homeostasis 

Overcoming suppressions (e.g., p53, immune, angiogenesis)

Key mechanisticconsideration

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, ……

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

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

(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

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.)

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

TCDD Ah Receptor Agonists SAR

- Rectangle 3 x 10 Angstroms- Planar molecule- Substitution at lateral positions

O

O

Cl

Cl Cl

Cl

Cl

Cl

Cl

Cl

ClCl

ClCl

Cl

ClCl

O

Cl

Cl Cl

Cl

Cl

2,3,7,8-TCDD

3,3',4,4',5'-PCB 2,3,4,7,8-PCDB2,2',4,4',5,5'-HCB

NTP TechReport No.

Chemical Agent NTP Bioassay Results in Female Rats OncoLogicPredictionEvidence* Target Organ(s) Incidence**

521 2,3,7,8-TCDD CE Liver/lung/oral cavity 25/53 H

520 3,3’,4,4’,5’-PCB CE Liver/lung/oral cavity 13/53 HM

525 2,3,4,7,8-PCDB SE Liver/oral cavity 4/53 M

529 2,2’,4,4’,5,5’-HCB EE Liver 2/53 LM *CE = clear evidence; SE = some evidence; EE = equivocal evidence **Highest incidence observed in any one specific target organ.

Peroxisome Proliferator-Activated Receptor (PPARα) SAR

Relative peroxisome proliferative activity of chlorinated phenoxyacetic acid in cultured hepatocytes

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

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

Hormonal imbalance: basic principles

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?)

Some SA for thyroid carcinogens

NN

S

R'

H

R"

R

N,N'-Dicyclohexylthiourea (R=H; R'=R"=C6H11)N,N'-Diethylthiourea (R=H; R'=R"=C2H5) Trimethylthiourea (R=R'=R"=CH3)

NH NH

S

Ethylenethiourea

NH NH

S

R O

2-Thiouracil (R=H)

6-Methylthiouracil (R=CH3)

6-n-Propylthiouracil (R=C3H7)

NH-C(S)-SH

NH-C(S)-SH

Ethylenebis-dithiocarbamate

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

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

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

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

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)

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

Searching for a common mechanistic basis

Small tert-alcohol as potential SA formale rat kidney α2µnephropathy

CH3-CH-CH2-C-CH3

MIBK

CH3

CH3-C-CH2-C-CH3

O

OCH3

OH

CH3-CH-CH2-C-CH3

CH3

major metabolite of MIBK

CH3

Isooctane (TMP)

CH3-C-CH2-C-CH3

OH

CH3CH3

2-OH-TMP

CH3-C-CH3

CH3

O-CH3

CH3-C-CH3

CH3

MTBE

t-Butyl alcohol

OH

CH3

CH3

CH3

CH3

CH3-CH-O-C-CH3

HOH2C

PG t-Butyl Ether

Cytotoxicity induced nasal tumors

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

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

Binding of Okadaic Acid and Microcystin-LRto Protein Phosphate 2A Core Enzyme

(from Xing et al., Cell 127, 341, 2006)

Integrative Approach

Woo et al. (1998)

Initiation(e.g., Electrophilicity, DNA

adduct, genotox, transgenic rodent models, ras, etc.)

Progression(e.g, immune

suppression, free radical, metastasis, angiogenesis, etc.)

Promotion(e.g, cell proliferation, apoptosis, gap junct.cyp, hormonal imbal,

gene expression, mitogenesis,

ppar, myc, etc.)

EPA gene expression and pathway analysis study of nongenotoxic carcinogenic conazole pesticides

(from Hester et al. Tox. Sci. 127, 54, 2012)

N

N

NN

N

NN

N

N

OH Cl

O

F

Cl O

O

ClCl

Cyproconazole Epoxiconazole Propiconazole

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

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

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

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